Phosphoroamidate esters, and use and synthesis thereof

ABSTRACT

Phosphoramidate esters and related nucleotide analogs useful in polynucleotide sequencing techniques, and synthetic methods for preparing those compounds, are disclosed. These compounds include nucleotide phosphoramidates analogs that are modified on the alpha-phosphate to enable attachment of a variety of application-specific substituents such as tether molecules.

FIELD OF THE INVENTION

The present invention is in the field of phosphoroamidate estercompounds, synthetic methods for making said compounds, and methods forthe determination of nucleic acids using said compounds, e.g., in thefield of single molecule sequencing.

BACKGROUND

Chemically modified nucleotides have been extensively used in the studyof many complicated biological systems. In particular, they have provenindispensable in the analysis of protein-nucleic acid interactions, thedetermination of genotypes, and the sequencing of nucleic acids. Ingeneral, these applications rely on differences in the chemicalreactivity or electronic properties of the modified nucleotides ascompared to the naturally occurring counterpart. Analogs of nucleotidetriphosphates (NTPs) may be synthesized with modifications at the base,sugar, or triphosphate chain. Historically, modification of thetriphosphate chain has been mainly used to study enzymatic pathways,which results in hydrolysis and transfer of the phosphate from NTP toanother molecule. Modification of sugar and base has served a number ofdifferent purposes, from pharmaceutical to diagnostic applications.

In many examples of deoxyribonucleotide triphosphate (dNTP) analogs, theoriginal P¹—O—CH₂(5′) fragment has been modified. One common type ofmodification is the substitution of an oxygen atom with S, NH, or CR²(where R is H, an alkyl, or an aryl group and their derivatives).Interest in 5′-NH₂-dNTPs (i.e. 5′ phosphoramidates and analogs), inparticular, has increased due to their potential utility in genomicanalysis (see, e.g., Shchepinov et al., Matrix-induced fragmentation ofP3′-N5′ phosphoramidate-containing DNA: high-throughput MALDI-TOFanalysis of genomic sequence polymorphisms. Nuc. Acids Res. v. 30(17)pp. 3739-3747 (2002)) and DNA sequencing (see, e.g., U.S. Pat. No.8,324,360 to Kokoris et al.). Certain useful features of 5′phosphoramidate analogs include their ability to exist in a triphosphateform, which can be utilized by many polymerases (see, e.g., Letsinger etal., Incorporation of 5′-amino-5′-deoxythymidine 5′ phosphate inpolynucleotides by use of DNA polymerase I and a phiX174 DNA template.Biochemistry v. 15 pp. 2810-2816 (1975)), and the ability to selectivelycleave the P—N bond under acidic conditions (see, e.g., Letsinger etal., Enzymatic synthesis of polydeoxyribonucleotides possessinginternucleotide phosphoramidate bonds. J. Am. Chem. Soc. v. 94 pp.292-293 (1972)).

Modification of the dNTP alpha phosphate, in turn, has been exploited tointroduce diverse functional properties, such as attachment points fordetectable labels, solid state matrices, and other useful substituents.Examples of nucleotide analogs modified on the phosphate residue andvarious processes for producing such analogs have been described inseveral reviews. See, e.g., Koukhareva, Vaghefi and Lebedev, NucleosideTriphosphates and their Analogs (2005) Chapter 2, “Synthesis andproperties of NTP analogs with modified Triphosphate side chains”, Ed.M. Vaghefi, CRC Press, Taylor & Francis, Boca Raton. Triphosphates areof particular importance as substrates for DNA or RNA polymerase thatincorporate the nucleotide analogs into long chain nucleic acids.Generally, triphosphates are synthesized by first preparing thenucleoside monophosphates, which are subsequently converted intotriphosphates enzymatically, for example, by kinases. However,nucleotide monophosphate (NMP) analogs may not be suitable substratesfor kinase enzymes, and the preparation of such specific analogs thuswill likely require unique chemical routes.

Though a variety of different analogs are available that mimicnucleotides and their polymers for diverse applications, there remains aneed in the art for the development of novel analogs that offer uniquecombinations of individual properties while retaining the ability to berecognized and acted upon by enzymes. For example, the concept of“sequencing by expansion” has been described, in which a templatenucleic acid is converted into an expandable daughter-strand polymeric“surrogate” through template-directed enzymatic synthesis. In oneembodiment, the synthesis reaction incorporates dNTP analogs, referredto as “XNTPs” (see, e.g. Kokoris et al., U.S. Pat. No. 8,324,360). Onceincorporated into the surrogate, cleavage of the selectively cleavablebonds can effectively expand the polymer, thus increasing the spatialresolution of the individual nucleotides. Such expanded nucleic acidmolecules show great promise in, e.g., nanopore-based sequencingsystems. For this particular application, it would be advantageous toprovide improved polymerase substrate analogs that feature both aselectively cleavable bond and an attachment point for a bulkysubstituent, such that these features are introduced into the expandablesurrogate daughter-strand product.

Thus, one technical object forming the basis of the present invention isto provide improved nucleotide phosphoramidate analogs that are furthermodified on the alpha-phosphate to enable attachment of a variety ofapplication-specific substituents (e.g. tether molecules) and,furthermore, to provide reliable processes for the synthesis of suchnovel nucleotide analogs.

All of the subject matter discussed in the Background section is notnecessarily prior art and should not be assumed to be prior art merelyas a result of its discussion in the Background section. Along theselines, any recognition of problems in the prior art discussed in theBackground section or associated with such subject matter should not betreated as prior art unless expressly stated to be prior art. Instead,the discussion of any subject matter in the Background section should betreated as part of the inventor's approach to the particular problem,which in and of itself may also be inventive.

BRIEF SUMMARY

Briefly stated, the present disclosure provides mono andpolyphosphoroamidate ester compounds, synthetic methods for thepreparation of such compounds, compounds useful in the syntheticmethods, and uses for the compounds.

For example, in one aspect the present disclosure provides compounds ofthe formula (1)

wherein,

R¹ is selected from

-   -   a) an alkyl group and an oxyalkyl group, either of which        terminates in a functional group selected from carbon-carbon        double bond, carbon-carbon triple bond, hydroxyl, amine, azide,        hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;        and    -   b) an alkyl group and an oxyalkyl group, either of which        terminates in a linker group (LG1), the LG1 bonded to a tether        (T);

R² is selected from hydrogen and C₁-C₄alkyl;

R³ is selected from R⁵ and —[Pn-O]_(m)—R⁵, where Pn is independentlyselected from P(OR⁵) and P(═O)(OR⁵) at each occurrence, and m isselected from 1, 2, 3, 4, 5 and 6;

R⁴ is selected from

R⁵ is selected from H and G¹;

R⁶ is a heterocycle, the heterocycle optionally comprising a substituentR¹³, where R¹³ is selected from

-   -   a) an alkyl group and an oxyalkyl group, either of which        terminates in a functional group selected from carbon-carbon        double bond, carbon-carbon triple bond, hydroxyl, amine, azide,        hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;        and    -   b) an alkyl group and an oxyalkyl group, either of which        terminates in a linker group (LG2), the LG2 bonded to the tether        (T);

R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R² is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom;

G² is selected from oxygen, sulfur and CH₂; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

In another aspect, the present disclosure provides synthetic methods andcompounds useful therein, for the preparation of mono andpolyphosphoroamidate ester compounds. For example, the presentdisclosure provides a process for forming a phosphoromonoamidate diester110 comprising contacting compound 100 with compound 105 to providecompound 110, the contacting conducted in the presence of a halide anionsource,

wherein:

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen and C₁-C₄alkyl;

R⁴ is selected from

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R² is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom;

G² is selected from oxygen, sulfur and CH₂; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

As another example, the present disclosure provides a process of forminga phosphate protected N-phosphoroamidate-monoester diphosphate 120comprising contacting a compound 110 with a compound 115 followed byoxidation to provide compound 120,

wherein:

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen and C₁-C₄alkyl;

R⁴ is selected from

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R⁷ is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom;

G² is selected from oxygen, sulfur and CH₂; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

As another example, the present disclosure provides a process of forminga phosphate protected N-phosphoroamidate-monoester triphosphate (125)comprising contacting a compound (120) with a compound (115) to provide,after oxidation, a phosphate protected N-phosphoroamidate-monoestertriphosphate (125),

wherein:

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen and C₁-C₄alkyl;

R⁴ is selected from

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R⁷ is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom;

G² is selected from oxygen, sulfur and CH₂; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

As an example of a compound useful in the synthetic methods, in oneembodiment the present disclosure provides a cyclic phosphite of theformula

wherein R¹ is an alkyl group or an oxyalkyl group, either of which isterminally-functionalized, where the terminal functional group isselected from carbon-carbon double bond, carbon-carbon triple bond,hydroxyl, amine, azide, hydrazine, thiol, carboxyl or ester thereof,formyl, hydroxylamino and halogen. For example, in individualembodiments, the terminal functional group of R¹ may be carbon-carbondouble bond; and/or it may be carbon-carbon triple bond; and/or it maybe hydroxyl; and/or it may be amine; and/or it may be thiol; and/or itmay be carboxyl or ester thereof; and/or it may be formyl; and/or it maybe hydroxylamino; and/or it may be halogen. In one embodiment, R¹comprises an alkyl group. For example, when R¹ is an alkyl group and thefunctional group is a carbon-carbon triple bond, R¹ may be—(CH₂)_(q)—C≡CH where —(CH₂)_(q) is the alkyl group, which might also bereferred to as an alkylene group, and q is an integer selected from2-10, e.g., R¹ is 1-hexynyl of the formula —CH₂CH₂CH₂CH₂C≡CH. In oneembodiment, R¹ includes an electrophilic group. In one embodiment, R¹includes a nucleophilic group. In one embodiment, R¹ includes acarboxylic acid or an ester thereof. In one embodiment, R¹ is an alkylgroup which is terminally-functionalized. In one embodiment, R¹ is anoxyalkyl group which is terminally functionalized, where an oxyalkylgroup may also be called an oxyalkylene group, and refers to an alkylgroup that incorporates one or more oxygen atoms in the form of ethergroups. Oxyethylene (—O—CH₂—CH₂—) groups and oxypropylene(—O—CH₂—CH₂—CH₂—) groups are exemplary oxyalkyl groups. The oxyalkylgroup of R¹ may be formed from one or a plurality of oxyalkyl units,such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 repeating units.

In one embodiment of the present disclosure, the cyclic phosphite asdescribed herein may be used in a process for forming aN-phosphoroamidate-monoester triphosphate (160) from thecyclotriphosphite (155) and an azide (105)

the process comprising combining (155) and (105) in the presence ofsuitable solvent and at a suitable temperature for a suitable reactionperiod, so as to form (160), wherein:

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁴ is selected from

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R⁷ is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G² is selected from oxygen, sulfur and CH₂; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

This Brief Summary has been provided to introduce certain concepts in asimplified form that are further described in detail below in theDetailed Description. Except where otherwise expressly stated, thisBrief Summary is not intended to identify key or essential features ofthe claimed subject matter, nor is it intended to limit the scope of theclaimed subject matter.

The details of one or more embodiments are set forth in the descriptionbelow. The features illustrated or described in connection with oneexemplary embodiment may be combined with the features of otherembodiments. Thus, any of the various embodiments described herein canbe combined to provide further embodiments. Aspects of the embodimentscan be modified, if necessary to employ concepts of the various patents,applications and publications as identified herein to provide yetfurther embodiments. Other features, objects and advantages will beapparent from the description, the drawings, and the claims.

BRIEF DESCRIPTION OF THE DRAWING

The FIGURE illustrates how a nucleobase triphosphoramidate of thepresent disclosure may function as a component of a XNTP substrateuseful in Sequencing by Expansion (SBX).

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to one or more of new compounds,methods for preparing compounds including novel compounds useful in thesynthetic methods, and the use of these compounds in, for example,nucleic acid sequencing techniques, each as disclosed herein. Prior tosetting forth this disclosure in more detail, it may be helpful to anunderstanding thereof to provide definitions of certain terms to be usedherein. Additional definitions are set forth throughout this disclosure.

“Independently at each occurrence” means that whenever a particularvariable occurs, and that variable may be selected from two or moreoptions, then at each occurrence of that variable, any of the two ormore options may be selected, regardless of the selection made at anyother occurrence of the variable. For example, when Pn is selected from—P(OR⁵) and —P(═O)(OR⁵) where m is selected from 2, 3, 4, 5 and 6, thenat each of the 2-6 occurrences of Pn, Pn may represent —P(OR⁵) or—P(═O)(OR⁵), and R⁵ is likewise independently selected at eachoccurrence. Unless otherwise specified, when a variable may be selectedmore than once in a formula, each selection is made independent at eachoccurrence of the variable.

Alkyl groups include straight chain and branched alkyl groups andcycloalkyl groups having from 1 to about 20 carbon atoms (C₁-C₂₀ alkylor C₁₋₂₀ alkyl), and typically from 1 to 12 carbons (C₁-C₁₂ alkyl orC₁₋₁₂ alkyl) or, in some embodiments, from 1 to 8 carbon atoms (C₁-C₈alkyl or C₁₋₈ alkyl) or, in some embodiments, from 1 to 4 carbon atoms(C₁-C₄ alkyl or C₁₋₄ alkyl) or, in some embodiments, from 1 to 3 carbonatoms (C₁-C₃ alkyl or C₁₋₃ alkyl). Examples of straight chain alkylgroups include, but are not limited to, methyl, ethyl, n-propyl,n-butyl, n-pentyl, n-hexyl, n-heptyl, and n-octyl groups. Examples ofbranched alkyl groups include, but are not limited to, isopropyl,iso-butyl, sec-butyl, t-butyl, neopentyl, isopentyl, and2,2-dimethylpropyl groups. The alkyl group may be substituted orotherwise functionalized with a functional group. Representativesubstituted alkyl groups can be substituted one or more times with anynon-alkyl group, for example, amino, hydroxy, cyano, carboxy, nitro,thio, alkoxy, and halogen groups. The alkyl group may contain one ormore carbon-carbon double bonds and one or more carbon-carbon triplebonds along its structure. The term “terminally functionalized alkylgroup” and its equivalent term “omega-functionalized alkyl group” refersto an alkyl group that terminates in a functional group. For example,the group —CH₂CH₂CH₂CH₂—OH represents a terminally functionalizedn-butyl group having hydroxyl as the functional group, where this groupmay also be described as n-hydroxy C₄ alkyl.

Unsubstituted alkyl groups, which are optionally functionalized by thepresence of carbon-carbon double bonds and/or carbon-carbon triplebonds, are examples of hydrocarbon groups, i.e., groups formed entirelyof carbon and hydrogen. In one embodiment, the hydrocarbon group is analkyl group. A C₆-C₁₆ hydrocarbon has 6, 7, 8, 9, 10, 11, 12, 13, 14, 15or 16 carbon atoms in addition to hydrogen atoms as the only atomspresent in the hydrocarbon moiety. As disclosed elsewhere herein, eachof R¹ and R¹³ may be a hydrocarbon alkyl group. As also disclosedelsewhere herein, each of R¹ and R¹³ may be an oxyalkyl group.

Oxyalkyl refers to alkyl groups that are separated by oxygen, i.e.,alkyl-O-alkyl- etc. and the like. Exemplary alkyl groups in an oxyalkylunit are ethyl and propyl. Using ethyl as an example, oxyalkyl may referto one or more repeating units of —CH₂—CH₂—O—. Thus,-ethyl-O-ethyl-O-ethyl-O-ethyl is an exemplary oxyalkyl group. Thenumber of repeating alkyl-O units in an oxyalkyl may be, for example, 2,3, 4, 5, 6, 7, 8, 9, 10, or more than 10.

Halogen refers to bromide, chloride, iodide and fluoride.

In the structures shown herein, when not all natural valencies of anatom are filled by named groups, it should be understood that theunfilled valencies are filled by hydrogen. For example, the structuredrawn as

is equivalently drawn as

When a wavy line

in a chemical moiety intersects a bond, then the intersected bond is thelocation where the chemical moiety joins to the remainder of themolecule. All chiral, diastereomeric, racemic forms of a structure areintended, unless a particular stereochemistry or isomeric form isspecifically indicated. Compounds used in the present invention caninclude enriched or resolved optical isomers at any or all asymmetricatoms as are apparent from the depictions, at any degree of enrichment.Both racemic and diastereomeric mixtures, as well as the individualoptical isomers can be synthesized so as to be substantially free oftheir enantiomeric or diastereomeric partners, and these are all withinthe scope of certain embodiments of the invention.

Heterocycle and heterocyclyl groups include aromatic and non-aromaticring compounds (heterocyclic rings) containing 3 or more ring members,of which one or more is a heteroatom such as, but not limited to, N, O,S, or P. In some embodiments, heterocyclyl groups include 3 to 20 ringmembers, whereas other such groups have 3 to 15 ring members. At leastone ring contains a heteroatom, but every ring in a heteropolycyclicsystem need not contain a heteroatom. For example, a dioxolanyl ring anda benzdioxolanyl ring system (methylenedioxyphenyl ring system) are bothheterocyclyl groups within the meaning herein. A heterocyclyl groupdesignated as a C₂-heterocyclyl can be a 5-membered ring with two carbonatoms and three heteroatoms, a 6-membered ring with two carbon atoms andfour heteroatoms and so forth. Likewise a C₄-heterocyclyl can be a5-membered ring with one heteroatom, a 6-membered ring with twoheteroatoms, and so forth. The number of carbon atoms plus the number ofheteroatoms sums up to equal the total number of ring atoms. A saturatedheterocyclic ring refers to a heterocyclic ring containing nounsaturated carbon atoms.

The phrases “heterocycle” and “heterocyclyl group” includes fused ringspecies including those having fused aromatic and non-aromatic groups.The phrase also includes polycyclic ring systems containing a heteroatomand also includes heterocyclyl groups that have substituents, includingbut not limited to alkyl, halo, amino, hydroxy, cyano, carboxy, nitro,thio, or alkoxy groups, bonded to one of the ring members. Aheterocyclyl group as defined herein can be a heteroaryl group or apartially or completely saturated cyclic group including at least onering heteroatom. Heterocyclyl groups include, but are not limited to,pyrrolidinyl, furanyl, tetrahydrofuranyl, dioxolanyl, piperidinyl,piperazinyl, morpholinyl, pyrrolyl, pyrazolyl, triazolyl, tetrazolyl,oxazolyl, isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl,benzofuranyl, dihydrobenzofuranyl, indolyl, dihydroindolyl, azaindolyl,indazolyl, benzimidazolyl, azabenzimidazolyl, benzoxazolyl,benzothiazolyl, benzothiadiazolyl, imidazopyridinyl, isoxazolopyridinyl,thianaphthalenyl, purinyl, xanthinyl, adeninyl, guaninyl, quinolinyl,iso quinolinyl, tetrahydroquinolinyl, quinoxalinyl, and quinazolinylgroups.

As mentioned above, heterocyclyl groups may be substituted.Representative substituted heterocyclyl groups can be mono-substitutedor substituted more than once, including but not limited to, ringscontaining at least one heteroatom which are mono, di, tri, tetra,penta, hexa, or higher-substituted with substituents such as thoselisted above, including but not limited to substituted alkyl where thesubstituent may be, for example, halo, amino, hydroxy, cyano, carboxy,azide, hydrazine, nitro, thio, or alkoxy; unsaturated alkyl having, forexample, carbon-carbon double bonds and/or carbon-carbon triple bonds;and alkyl groups that are both unsaturated and substituted.

Heteroaryl groups are aromatic ring compounds containing 5 or more ringmembers, of which, one or more is a heteroatom such as, but not limitedto, N, O, and S. A heteroaryl group designated as a C₂-heteroaryl can bea 5-membered ring with two carbon atoms and three heteroatoms, a6-membered ring with two carbon atoms and four heteroatoms and so forth.Likewise a C₄-heteroaryl can be a 5-membered ring with one heteroatom, a6-membered ring with two heteroatoms, and so forth. The number of carbonatoms plus the number of heteroatoms sums up to equal the total numberof ring atoms. Heteroaryl groups include, but are not limited to, groupssuch as pyrrolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl,isoxazolyl, thiazolyl, pyridinyl, thiophenyl, benzothiophenyl,benzofuranyl, indolyl, azaindolyl, indazolyl, benzimidazolyl,azabenzimidazolyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl,imidazopyridinyl, isoxazolopyridinyl, thianaphthalenyl, purinyl,xanthinyl, adeninyl, guaninyl, quinolinyl, iso quinolinyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, quinoxalinyl, andquinazolinyl groups.

The terms “heteroaryl” and “heteroaryl groups” include fused ringcompounds such as wherein at least one ring, but not necessarily allrings, are aromatic, including tetrahydroquinolinyl,tetrahydroisoquinolinyl, indolyl and 2,3-dihydro indolyl. The term alsoincludes heteroaryl groups that have other groups bonded to one of thering members, including but not limited to alkyl, halo, amino, hydroxy,cyano, carboxy, nitro, thio, or alkoxy groups. Representativesubstituted heteroaryl groups can be substituted one or more times withsubstituents such as those listed herein.

In one embodiment, the heterocycle group is a “nucleobase”, where thisterm refers to a heterocyclic base such as adenine, guanine, cytosine,thymine, uracil, inosine, xanthine, hypoxanthine, or a heterocyclicderivative, analog, or tautomer thereof. A nucleobase can be naturallyoccurring or synthetic. Non-limiting examples of nucleobases areadenine, guanine, thymine, cytosine, uracil, xanthine, hypoxanthine,8-azapurine, purines substituted at the 8 position with methyl orbromine, 9-oxo-N6-methyladenine, 2-aminoadenine, 7-deazaxanthine,7-deazaguanine, 7-deaza-adenine, N4-ethanocytosine, 2,6-diaminopurine,N6-ethano-2,6-diaminopurine, 5-methylcytosine,5-(C₃-C₆)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, thiouracil,pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine,isoguanine, inosine, 7,8-dimethylalloxazine, 6-dihydrothymine,5,6-dihydrouracil, 4-methyl-indole, ethenoadenine and the non-naturallyoccurring nucleobases described in U.S. Pat. Nos. 5,432,272 and6,150,510 and published PCT applications WO 92/002258, WO 93/10820, WO94/22892 and WO 94/24144, and Fasman (“Practical Handbook ofBiochemistry and Molecular Biology”, pp. 385-394, 1989, CRC Press, BocaRaton, La.), all herein incorporated by reference in their entireties.In one embodiment, the nucleobase is selected from adenine, guanine,uridine, and cytosine, and analogs of these nucleobases, such as thoseanalogs disclosed herein.

“Nucleobase residue” includes nucleotides, nucleosides, fragmentsthereof, and related molecules having the property of binding to acomplementary nucleotide. Deoxynucleotides and ribonucleotides, andtheir various analogs, are contemplated within the scope of thisdefinition. Nucleobase residues may be members of oligomers and probes.“Nucleobase” and “nucleobase residue” may be used interchangeably hereinand are generally synonymous unless context dictates otherwise.

In one embodiment, the heterocycle may be denoted by the symbol “B₁₋₄”,wherein the subscript indicates that the heterocycle may be any one ofthe four standard nucleobases, A, C, G, or T, or an analog thereofwherein one atom of a natural base is replaced with a different atomwhich typically allows for additional substitution on the nucleobase.

In one embodiment, the heterocycle is a heterocyclic base. Heterocyclicbases are well known in the art as being nitrogen containing ringstructures bound though a glycosidic bond to a sugar moiety, such as apentose (e.g., D-ribose and 2-deoxy-D-ribose), where the sugar moietymay be bonded to a phosphate, such as a monophosphate, a diphosphate anda triphosphate. Exemplary heterocyclic bases are purines andpyrimidines. Exemplary purines ae adenine and guanine, while exemplarypyrimidines are cytosine, uracil and thymine. The heterocyclic baseincludes substituted heterocyclic bases and analogs of a naturallyoccurring heterocyclic base wherein a native atom is replaced with adifferent atom (e.g., a nitrogen normally found in a heterocyclic basemay be replaced with carbon, e.g., C—H or C-substituent). See, e.g.,Nucleic acids in chemistry and biology. Edited by C. Michael Blackburnand Michael J. Gait, Oxford and New York: Oxford University Press, 1996,pp. xix+ 528.

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom. In one embodiment, G¹ is a protecting group for ahydroxyl group that is bonded to a phosphorous atom, or in other words,G¹ is a protecting group that is bonded to an oxygen, where the oxygenis bonded to a phosphorous, so that the protecting group is protectingwhat would otherwise be a hydroxyl group bonded to the phosphorous atom.G³ is a protecting group for a hydroxyl group that is bonded to acarbon. In other words, G³ is a protecting group that is bonded to anoxygen, where the oxygen is bonded to a carbon atoms, so that theprotecting group is protecting what would otherwise be a hydroxyl groupbonded to a carbon atom. Protecting groups can render chemicalfunctionality inert to specific reaction conditions and can be appendedto and removed from such functionality in a molecule withoutsubstantially damaging the remainder of the molecule. Practitioners inthe art would be familiar with suitable protecting groups for use in thesynthetic methods of the invention. See, e.g., Greene and Wuts,Protective Groups in Organic Synthesis, 2″ ed., John Wiley k Sons, NewYork, 1991 and Peter G. M. Wuts, “Greene's Protective Groups in OrganicSynthesis: Fifth Edition”, Wiley, 2014.

When a term refers to an integer selected from a range, then that termmay be any integer within that range, including the ends of the range.For example, when q is an integer selected from 2-10, then q can be anyof 2, 3, 4, 5, 6, 7, 8, 9 and 10.

SS represents a solid support such as controlled pore glass (CPG). SS-Lrepresents a solid support that is optionally bonded to a linking group,unless the presence of the linking groups is specifically excluded.Unless otherwise specified, a linking group is optionally insertedbetween a solid support and the compound being synthesized by solidphase chemistry as disclosed herein.

It is to be understood that the terminology used herein is for thepurpose of describing specific embodiments only and is not intended tobe limiting. It is further to be understood that unless specificallydefined herein, the terminology used herein is to be given itstraditional meaning as known in the relevant art.

Reference throughout this specification to “one embodiment” or “anembodiment” and variations thereof means that a particular feature,structure, or characteristic described in connection with the embodimentis included in at least one embodiment. Thus, the appearances of thephrases “in one embodiment” or “in an embodiment” in various placesthroughout this specification are not necessarily all referring to thesame embodiment. Furthermore, the particular features, structures, orcharacteristics may be combined in any suitable manner in one or moreembodiments.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents, i.e., one or more,unless the content and context clearly dictates otherwise. It shouldalso be noted that the conjunctive terms, “and” and “or” are generallyemployed in the broadest sense to include “and/or” unless the contentand context clearly dictates inclusivity or exclusivity as the case maybe. Thus, the use of the alternative (e.g., “or”) should be understoodto mean either one, both, or any combination thereof of thealternatives. In addition, the composition of “and” and “or” whenrecited herein as “and/or” is intended to encompass an embodiment thatincludes all of the associated items or ideas and one or more otheralternative embodiments that include fewer than all of the associateditems or ideas.

Unless the context requires otherwise, throughout the specification andclaims that follow, the word “comprise” and synonyms and variantsthereof such as “have” and “include”, as well as variations thereof suchas “comprises” and “comprising” are to be construed in an open,inclusive sense, e.g., “including, but not limited to.” The term“consisting essentially of” limits the scope of a claim to the specifiedmaterials or steps, or to those that do not materially affect the basicand novel characteristics of the claimed invention.

Any headings used within this document are only being utilized toexpedite its review by the reader, and should not be construed aslimiting the invention or claims in any manner. Thus, the headings andAbstract of the Disclosure provided herein are for convenience only anddo not interpret the scope or meaning of the embodiments.

In one embodiment, the present disclosure provides a compound of theformula (1)

wherein,

R¹ is selected from

-   -   a) an alkyl group and an oxyalkyl group, either of which        terminates in a functional group selected from carbon-carbon        double bond, carbon-carbon triple bond, hydroxyl, amine, azide,        hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;        and    -   b) an alkyl group and an oxyalkyl group, either of which        terminates in a linker group (LG1), the LG1 bonded to a tether        (T);

R² is selected from hydrogen and C₁-C₄alkyl;

R³ is selected from R⁵ and —[Pn-O]_(m)—R⁵, where Pn is independentlyselected from —P(OR⁵) and —P(═O)(OR⁵) at each occurrence, and m isselected from 1, 2, 3, 4, 5 and 6;

R⁴ is selected from

R⁵ is selected from H and G¹;

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from

-   -   a) an alkyl group and an oxyalkyl group, either of which        terminates in a functional group selected from carbon-carbon        double bond, carbon-carbon triple bond, hydroxyl, amine, azide,        hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;        and    -   b) an alkyl group and an oxyalkyl group, either of which        terminates in a linker group (LG2), the LG2 bonded to the tether        (T);

R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R² is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom;

G² is selected from oxygen, sulfur and CH₂; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

Unless otherwise specified, the following descriptions of R¹, R², etc.may be used to further describe any of the compounds and syntheticmethods disclosed herein which are described in terms of R¹, R², etc.

In one embodiment, the R¹ group is a terminally-functionalized alkylgroup, where the functional group is selected from, for example,carbon-carbon double bond, carbon-carbon triple bond, nucleophilicgroups such as hydroxyl, thiol or amino, electrophilic groups such ashalogen, or other reactive groups such as carboxyl, formyl (aldehyde)and hydroxyamino. In optional embodiments: the functional group iscarbon-carbon double bond; the functional group is carbon-carbon triplebond; the functional group is hydroxyl; the functional group is amine;the functional group is thiol; the functional group is halogen; thefunctional group is carboxyl or an ester thereof; the functional groupis formyl, also known as aldehyde (—C(═O)H); the functional group ishydroxylamine. In optional embodiments, the alkyl group is a straightchain or a branched alkyl group having from 1 to about 20 carbon atoms(C₁-C₂₀ alkyl or C₁₋₂₀ alkyl), or 1 to 12 carbons (C₁-C₁₂ alkyl or C₁₋₁₂alkyl) or 1 to 8 carbon atoms (C₁-C₈ alkyl or C₁₋₈ alkyl) or 1 to 4carbon atoms (C₁-C₄ alkyl or C₁₋₄ alkyl) or, in some embodiments, from 1to 3 carbon atoms (C₁-C₃ alkyl or C₁₋₃ alkyl). In one embodiment, R¹ isa hydrocarbon group such as —(CH₂)_(q)—C≡CH and q is an integer selectedfrom 2-10, e.g., R¹ may be 1-hexynyl of the formula —CH₂CH₂CH₂CH₂C≡CH.In one embodiment, R¹ includes an electrophilic group as part of itsstructure, preferably the electrophilic group being at the terminus ofthe R¹ group. In one embodiment, R¹ includes a nucleophilic group aspart of its structure, preferably the nucleophilic group being at theterminus of the R¹ group. In one embodiment, R¹ includes a carboxylicacid or an ester thereof as part of its structure, where the carboxylicacid or an ester thereof is preferably at the terminus of the R¹ group.

In another embodiment, R¹ is an oxyalkyl group which is terminallyfunctionalized, where an oxyalkyl group may also be called anoxyalkylene group, and refers to an alkyl group that incorporates one ormore oxygen atoms in the form of ether groups. For example, the oxyalkylgroup may be an oxyethyl (—O—CH₂—CH₂—) group or an oxypropyl(—O—CH₂—CH₂—CH₂—) group, those being are exemplary oxyalkyl groups. Theoxyalkyl group of R¹ may be formed from one or a plurality of oxyalkylunits, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 repeatingunits. The functional group is selected from, for example, carbon-carbondouble bond, carbon-carbon triple bond, nucleophilic groups such ashydroxyl, thiol or amino, electrophilic groups such as halogen, or otherreactive groups such as carboxyl, formyl (aldehyde) and hydroxyamino. Inoptional embodiments: the functional group is carbon-carbon double bond;the functional group is carbon-carbon triple bond; the functional groupis hydroxyl; the functional group is amine; the functional group isthiol; the functional group is halogen; the functional group is carboxylor an ester thereof; the functional group is formyl, also known asaldehyde (—C(═O)H); the functional group is hydroxylamine. In optionalembodiments, the alkyl portion of the oxyalkyl is a straight chain or abranched alkyl group having from 1 to about 20 carbon atoms (C₁-C₂₀alkyl or C₁₋₂₀ alkyl), or 1 to 12 carbons (C₁-C₁₂ alkyl or C₁₋₁₂ alkyl)or 1 to 8 carbon atoms (C₁-C₈ alkyl or C₁₋₈ alkyl) or 1 to 4 carbonatoms (C₁-C₄ alkyl or C₁₋₄ alkyl) or, in some embodiments, has 1, 2 or 3carbon atoms (C₁-C₃ alkyl or C₁₋₃ alkyl), or has 2 carbon atoms. In oneembodiment, R¹ includes an electrophilic group as part of its structure,preferably the electrophilic group being at the terminus of the regroup.In one embodiment, R¹ includes a nucleophilic group as part of itsstructure, preferably the nucleophilic group being at the terminus ofthe regroup. In one embodiment, R¹ includes a carboxylic acid or anester thereof as part of its structure, where the carboxylic acid or anester thereof is preferably at the terminus of the regroup.

Thus, R¹ may be an alkyl group or an oxyalkyl group, either of which isterminally-functionalized, where the terminal functional group isselected from carbon-carbon double bond, carbon-carbon triple bond,hydroxyl, amine, azide, hydrazine, thiol, carboxyl or ester thereof,formyl, hydroxylamino and halogen.

The R² group is selected from hydrogen and C₁-C₄alkyl. In oneembodiment, R² is hydrogen in each of the embodiments and embodimentcombinations as disclosed herein. In another embodiment, R² is C₁, i.e.,methyl.

The R³ group is selected from R⁵ and —[Pn-O]_(m)—R⁵, where the term Pnis used to refer to the two options —P(OR⁵) and —P(═O)(OR⁵), where thesetwo options are independently selected at each occurrence of Pn, and mis selected from 1, 2, 3, 4, 5 and 6. R⁵ is selected from H and G¹; andG¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom. In one embodiment, R³ represents R⁵, and R⁵ is H. Inanother embodiment, R³ represents R⁵, and R⁵ is a protecting group G¹.

In other embodiments, R³ is —[Pn-O]_(m)—R⁵. Thus, depending on theselections for —[Pn-O]_(m)—R⁵, the present disclosure provides each ofthe following exemplary structures (1A)-(1K):

Thus, the compounds of the present disclosure may have, for example,multiple phosphate groups or phosphate ester groups. In one embodiment,each of the phosphorous atoms is in the +5 valence state except for theterminal phosphorous atom which is in the +3 valence state, and R⁵ maybe hydrogen or G¹, independently selected at each occurrence. A fewexemplary structures of this type are shown below as structures(1L)-(1Q):

The R⁴ group is selected from

In one embodiment, R⁴ is a cyclic group of the formula

In another embodiment, the R⁴ group is an acyclic groups of the formula

In either of these embodiments, R⁶ is a heterocycle, where in optionalembodiments R⁶ is a nucleobase or a heterocyclic base that may besubstituted with R¹³ as defined herein.

Exemplary R⁶ nucleobases are the B₁₋₄ nucleobases, where this termrefers to a nucleobase selected from an adenosine analog, a guanosineanalog, a uridine analog and a cytidine analog. For example, B₁₋₄ mayrefer to an adenosine analog of formula

a guanosine analog of formula

a uridine analog of formula

or a cytidine analog of formula

wherein R¹³ is selected from an alkyl group and an oxyalkyl group,either of which is terminally functionalized. The term “either of which”as used herein refers to the alkyl group and the oxyalkyl group.Exemplary functional groups are selected from carbon-carbon double bond,carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol,carboxyl, formyl, hydroxylamino and halogen. In one embodiment, theterminal functional group of R¹³ and the terminal functional group of R¹are the same functional group, for example, the terminal functionalgroup of both R¹ and R¹³ is a carbon-carbon triple bond such as shown incompound (11) disclosed elsewhere herein.

Thus, the R¹³ group may be a terminally-functionalized alkyl group,where the functional group may be selected from carbon-carbon doublebond, carbon-carbon triple bond, nucleophilic groups such as hydroxyl,thiol or amino, electrophilic groups such as halogen, or other reactivegroups such as carboxyl, formyl (aldehyde) and hydroxyamino. In optionalembodiments: the functional group is carbon-carbon double bond; thefunctional group is carbon-carbon triple bond; the functional group ishydroxyl; the functional group is amine; the functional group is thiol;the functional group is halogen; the functional group is carboxyl or anester thereof; the functional group is formyl, also known as aldehyde(—C(═O)H); the functional group is hydroxylamine. In optionalembodiments, the alkyl group is a straight chain or a branched alkylgroup having from 1 to about 20 carbon atoms (C₁-C₂₀ alkyl or C₁₋₂₀alkyl), or 1 to 12 carbons (C₁-C₁₂ alkyl or C₁₋₁₂ alkyl) or 1 to 8carbon atoms (C₁-C₈ alkyl or C₁₋₈ alkyl) or 1 to 4 carbon atoms (C₁-C₄alkyl or C₁₋₄ alkyl) or, in some embodiments, from 1 to 3 carbon atoms(C₁-C₃ alkyl or C₁₋₃ alkyl). In one embodiment, R¹ is a hydrocarbonalkyl group that is or includes the moiety —(CH₂)_(r)—C≡CH and r is aninteger selected from 2-10, e.g., R¹³ is or comprises 1-hexynyl of theformula —CH₂CH₂CH₂CH₂C≡CH. In one embodiment, R¹³ includes anelectrophilic group as part of its structure, preferably theelectrophilic group being at the terminus of the R¹³ group. In oneembodiment, R¹³ includes a nucleophilic group as part of its structure,preferably the nucleophilic group being at the terminus of the R¹³group. In one embodiment, R¹³ includes a carboxylic acid or an esterthereof as part of its structure, where the carboxylic acid or an esterthereof is preferably at the terminus of the R¹³ group. As mentionedelsewhere herein, in one embodiment the terminal reactive group of R¹ isidentical to the terminal reactive group of R¹³, e.g., both R¹ and R¹³terminate in a carbon-carbon triple bond, e.g., each may terminate in a—CH₂—CH₂—CH₂—CH₂—C≡CH group. In one embodiment, R¹³ is or comprises anomega-functionalized C₆-C₁₆ hydrocarbon or an omega-functionalizedC₆-C₁₆alkyl.

However, in another embodiment, the R¹³ group may be aterminally-functionalized oxyalkyl group, where the functional group maybe selected from carbon-carbon double bond, carbon-carbon triple bond,nucleophilic groups such as hydroxyl, thiol or amino, electrophilicgroups such as halogen, or other reactive groups such as carboxyl,formyl (aldehyde) and hydroxyamino. In optional embodiments: thefunctional group is carbon-carbon double bond; the functional group iscarbon-carbon triple bond; the functional group is hydroxyl; thefunctional group is amine; the functional group is thiol; the functionalgroup is halogen; the functional group is carboxyl or an ester thereof;the functional group is formyl, also known as aldehyde (—C(═O)H); thefunctional group is hydroxylamine. In optional embodiments, the oxyalkylgroup incorporates a straight chain or a branched alkyl group havingfrom 1 to about 20 carbon atoms (C₁-C₂₀ alkyl or C₁₋₂₀ alkyl), or 1 to12 carbons (C₁-C₁₂ alkyl or C₁₋₁₂ alkyl) or 1 to 8 carbon atoms (C₁-C₈alkyl or C₁₋₈ alkyl) or 1 to 4 carbon atoms (C₁-C₄ alkyl or C₁₋₄ alkyl)or, in some embodiments, from 1 to 3 carbon atoms (C₁-C₃ alkyl or C₁₋₃alkyl), while in one embodiment the alkyl group of each oxyalkyl unithas 2 carbons, and in another embodiment the alkyl group of eachoxyalkyl unit has 3 carbons. In one embodiment, R¹³ includes anelectrophilic group as part of its structure, preferably theelectrophilic group being at the terminus of the R¹³ group. In oneembodiment, R¹³ includes a nucleophilic group as part of its structure,preferably the nucleophilic group being at the terminus of the R¹³group. In one embodiment, R¹³ includes a carboxylic acid or an esterthereof as part of its structure, where the carboxylic acid or an esterthereof is preferably at the terminus of the R¹³ group. As mentionedelsewhere herein, in one embodiment the terminal reactive group of R¹ isidentical to the terminal reactive group of R¹³, e.g., both R¹ and R¹³terminate in a carbon-carbon triple bond.

In one embodiment, R⁴ is a heterocycle or nucleobase which includes R¹³as part of its structure, where R¹³ is selected fromomega-functionalized C₆-C₁₆ hydrocarbons or omega-functionalized C₆-C₁₆alkyls. An exemplary R¹³ group is —C≡C—(CH₂)₄—C≡CH in which case theomega functional group is a carbon-carbon triple bond.

Thus, in another embodiment, R⁴ is a heterocycle or nucleobase whichincludes R¹³ as a substituent, where R¹³ is an alkyl group having aterminal carbon-carbon triple bond, and R¹ is an alkyl group having aterminal carbon-carbon triple bond, so that the compound of formula (1)may be a bis-alkyne deoxynucleoside polyphosphoramidate, e.g., abis-alkyne deoxynucleoside triphosphoramidate. Such a bis-alkynestructure is a particularly useful compound to react with a tetherprecursor having terminal azide groups, i.e., N₃-tether-N₃, where theproduct of such a reaction comprises triazole groups that link the twoends of the tether (via LG1 and LG2, each of LG1 and LG2 being atriazole group) to a deoxynucleoside polyphosphoramidate such as adeoxynucleoside triphosphoramidate.

In one embodiment, R¹³ is selected from omega-functionalized C₆-C₁₆hydrocarbons or omega-functionalized C₆-C₁₆ alkyls. An exemplary R¹³group is —C≡C—(CH₂)₄—C≡CH in which case the omega functional group is acarbon-carbon triple bond.

The R⁷ group is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl,hydroxyl and —CH₂—Or¹⁰. In various embodiments: R⁷ hydrogen; R⁷ is—CH₂-halogen; R⁷ is C₁-C₄alkyl; R₇ is hydroxyl and/or R⁷ is —CH²—OR¹⁰.In one embodiment, R⁷ is selected from hydrogen, —CH₂-halogen,C₁-C₄alkyl and —CH₂—OR¹⁰.

The R⁸ group is —OR¹¹ or —O-SSL. SSL and SS-L designate a solid support(SS) that is optionally bound to a linking (also referred to as alinker) group (L), where the linker group joins the solid supportthrough an oxygen atom as shown, to the remainder of the molecule.

The R⁹ group is hydrogen or, when R⁷ is —CH₂—OR¹⁰ then R⁹ may be—CH₂—R¹² where R¹⁰ and R¹² form a direct bond. In one embodiment, R⁹ ishydrogen. In one embodiment, R⁷ is —CH₂—OR¹⁰ and R⁹ is —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond.

The R¹¹ group is H or a protecting group G³; where G³ is a protectinggroup for a hydroxyl group that is bonded to a carbon atom. In oneembodiment, R¹¹ is hydrogen. In another embodiment, R¹¹ is a protectinggroup, G³.

The G² group is selected from oxygen, sulfur and CH₂. In one embodiment,G² is oxygen. In one embodiment, G² is sulfur. In one embodiment, G² isCH₂.

In one embodiment, the present disclosure provides a compound of theformula

wherein R¹, R², R³ and R⁴ are defined herein, and embodiments for R¹,R², R³ and R⁴ are provided, including embodiments for R⁵, R⁶, R⁷, R⁸,R⁹, R¹⁰, R¹¹, R¹² and R¹³, G¹, G², G³, Pn, m, d and r. In describingcompound of formula (I), any two, or any three, or any four, or anyfive, or more than five of these various embodiments may be combined.

For example, in one embodiment the present disclosure provides acompound of the formula (2a)

wherein

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen and C₁-C₄alkyl;

R⁵ is selected from H and G¹;

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R² is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom;

G² is selected from oxygen, sulfur and CH₂; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

In optional embodiments of compounds of formula (2a), R² is hydrogen;and independently G² is oxygen. For example, the present disclosureprovides a compound of formula (2b)

wherein

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁵ is selected from H and G¹;

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R⁷ is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

In another optional embodiment of compounds of formula (2a), R⁹ ishydrogen. In addition, G² is oxygen and/or R² is hydrogen. For example,the present disclosure provides a compound of formula (2c)

wherein

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁵ is selected from H and G¹;

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

The present disclosure also provides compounds corresponding to formulae(2a), (2b) and (2c) however the pentavalent phosphorous atom istrivalent. In other words, the present disclosure provides compounds offormula (2d), (2e) and (2f) where groups R¹, etc. are as defined above.

In specific embodiments of each of the compounds of formula (2a), (2b),(2c), (2d), (2e) and (2f): R¹ is a terminally-functionalized alkylgroup, where the functional group is carbon-carbon triple bond, e.g., R¹is —(CH₂)_(q)—C≡C and q is an integer selected from 2-10; and/or R⁵ inat least one occurrence is hydrogen, and/or R⁵ in at least oneoccurrence is G¹; and/or R⁶ is a nucleobase or R⁶ is a heterocyclicbase; and/or R⁷ is hydrogen; and/or R⁸ is OH or R⁸ comprises a solidsupport.

For example, in one embodiment the present disclosure provides acompound of the formula (3a)

wherein

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen and C₁-C₄alkyl;

R⁵ is selected from H and G¹;

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl and —CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R² is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom;

G² is selected from oxygen, sulfur and CH₂; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

In optional embodiments of compounds of formula (3a), R² is hydrogen;and independently G² is oxygen. For example, the present disclosureprovides compound of formula (3b)

wherein

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁵ is selected from H and G¹;

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R² is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

In another optional embodiment of compounds of formula (3a), R⁹ ishydrogen. In addition, G² is oxygen and/or R² is hydrogen. For example,the present disclosure provides a compound of formula (3c)

wherein

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁵ is selected from H and G¹;

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁷ is selected from hydrogen, —CH₂-halogen, hydroxyl and C₁-C₄alkyl;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

The present disclosure also provides compounds corresponding to formulae(3a), (3b) and (3c) however some of the pentavalent phosphorous atomsare trivalent. For example, the present disclosure provides compounds offormula (3d), (3e) and (3f) where groups R¹, etc. are as defined above.

In specific embodiments of each of the compounds of formula (3a), (3b),(3c), (3d), (3e) and (3f): R¹ is a terminally-functionalized alkylgroup, where the functional group is carbon-carbon triple bond, e.g., R¹is —(CH₂)_(q)—C≡C and q is an integer selected from 2-10; and/or

G² is oxygen; and/or R⁵ in at least one occurrence is hydrogen, and/orR⁵ in at least one occurrence is G¹; and/or R⁶ is a nucleobase or R⁶ isa heterocyclic base; and/or R⁷ is hydrogen; and/or R⁸ is OH or R⁸comprises a solid support. As another example, in one embodiment thepresent disclosure provides a compound of the formula (4a)

wherein: G¹ is H or a protecting group; R⁶ is a heterocycle; R⁸ isselected from OR¹¹ and O—SS; R¹¹ is selected from H and G³; G³ is aprotecting group for a hydroxyl group that is bonded to a carbon atom;and SS represents a solid support optionally bound to the O of O—SS viaa linking group (L). In one embodiment, R⁸ is hydroxyl. In oneembodiment, R⁶ is a nucleobase. In one embodiment, R⁶ is a heterocyclicbase. Optionally, the protecting group G¹ may be a 2-cyanoethyl group,giving rise to a compound of the present disclosure having the formula(4b)

wherein: R⁶ is a heterocycle; R⁸ is —OR¹¹ or —O-L-SS where L-SSrepresents a solid support optionally bound to a linker; R¹¹ is selectedfrom H and G³; G³ is a protecting group for a hydroxyl group that isbonded to a carbon atom; and SS represents a solid support optionallybound to the O of O—SS via a linking group (L). In one embodiment, R⁸ ishydroxyl. In one embodiment, R⁸ is protected hydroxyl. In yet anotherembodiment, R⁸ includes a solid support.

Optionally, R⁶ is a nucleobase. For example, in one embodiment R⁶ is auridine analog. An exemplary uridine analog is shown in the compound ofthe formula (4c),

wherein: G¹ is shown as cyanoethyl however other protecting groups maybe substituted for cyanoethyl; R¹³ is shown as —C≡C—(CH₂)₄—C≡CH howeverother omega-functionalized C₆-C₁₆ hydrocarbon groups may be substitutedfor —C≡C—(CH₂)₄—C≡CH; R⁸ is —OR¹¹ or —O-L-SS where L-SS represents asolid support optionally bound to a linker; R¹¹ is selected from H andG³; G³ is a protecting group for a hydroxyl group that is bonded to acarbon atom; and SS represents a solid support optionally bound to the Oof O—SS via a linking group. In one embodiment, R⁸ is hydroxyl. In oneembodiment, R⁸ is protected hydroxyl. In yet another embodiment, R⁸includes a solid support. As another example, in one embodiment R⁶ is acytidine analog. An exemplary cytidine analog is shown in the compoundof the formula (4d),

wherein: G¹ is shown as cyanoethyl however other protecting groups maybe substituted for cyanoethyl; R¹³ is shown as —C≡C—(CH₂)₄—C≡CH howeverother omega-functionalized C₆-C₁₆ hydrocarbon groups may be substitutedfor —C≡C—(CH₂)₄—C≡CH; R⁸ is —OR¹¹ or —O-L-SS where L-SS represents asolid support optionally bound to a linker; R¹¹ is selected from H andG³; and G³ is a protecting group for a hydroxyl group that is bonded toa carbon atom. In one embodiment, R⁸ is hydroxyl. In one embodiment, R⁸is protected hydroxyl. In yet another embodiment, R⁸ includes a solidsupport. As another example, in one embodiment R⁶ is an adenosineanalog. An exemplary adenosine analog is shown in the compound of theformula (4e),

wherein: G¹ is shown as cyanoethyl however other protecting groups maybe substituted for cyanoethyl; R¹³ is shown as —C≡C—(CH₂)₄—C≡CH howeverother omega-functionalized C₆-C₁₆ hydrocarbon groups may be substitutedfor —C≡C—(CH₂)₄—C≡CH; R⁸ is —OR¹¹ or —O-L-SS where L-SS represents asolid support optionally bound to a linker; R¹¹ is selected from H andG³; and G³ is a protecting group for a hydroxyl group that is bonded toa carbon atom. In one embodiment, R⁸ is hydroxyl. In one embodiment, R⁸is protected hydroxyl. In yet another embodiment, R⁸ includes a solidsupport. As a further example, in one embodiment R⁶ is a guanosineanalog. An exemplary guanosine analog is shown in the compound of theformula (4f),

wherein: G¹ is shown as cyanoethyl however other protecting groups maybe substituted for cyanoethyl; R¹³ is shown as —C≡C—(CH₂)₄—C≡CH howeverother omega-functionalized C₆-C₁₆ hydrocarbon groups may be substitutedfor —C≡C—(CH₂)₄—C≡CH; R⁸ is —OR¹¹ or —O-L-SS where L-SS represents asolid support optionally bound to a linker; R¹¹ is selected from H andG³; and G³ is a protecting group for a hydroxyl group that is bonded toa carbon atom. In one embodiment, R⁸ is hydroxyl. In one embodiment, R⁸is protected hydroxyl. In yet another embodiment, R⁸ includes a solidsupport.

The present disclosure also provides a cyclic phosphite of the formula(5) and salts thereof,

wherein R¹ is an alkyl group or an oxyalkyl group, either of which isterminally-functionalized, where the terminal functional group isselected from carbon-carbon double bond, carbon-carbon triple bond,hydroxyl, amine, azide, hydrazine, thiol, carboxyl or ester thereof,formyl, hydroxylamino and halogen. For example, in individualembodiments, the terminal functional group of R¹ may be carbon-carbondouble bond; and/or it may be carbon-carbon triple bond; and/or it maybe hydroxyl; and/or it may be amine; and/or it may be thiol; and/or itmay be carboxyl or ester thereof; and/or it may be formyl; and/or it maybe hydroxylamino; and/or it may be halogen. For example, when R¹ is analkyl group and the functional group is a carbon-carbon triple bond, R¹may be —(CH₂)_(q)—C≡CH where —(CH₂)_(q) is the alkyl group, which mightalso be referred to as an alkylene group, and q is an integer selectedfrom 2-10, e.g., R¹ is 1-hexynyl of the formula —CH₂CH₂CH₂CH₂C≡CH. Inone embodiment, R¹ includes an electrophilic group. In one embodiment,R¹ includes a nucleophilic group. In one embodiment, R¹ includes acarboxylic acid or an ester thereof. In one embodiment, R¹ is an alkylgroup which is terminally-functionalized. In one embodiment, R¹ is anoxyalkyl group which is terminally functionalized, where an oxyalkylgroup may also be called an oxyalkylene group, and refers to an alkylgroup that incorporates one or more oxygen atoms in the form of ethergroups. Oxyethyl (—O—CH₂—CH₂—) groups and oxypropyl (—O—CH₂—CH₂—CH₂—)groups are exemplary oxyalkyl groups. The oxyalkyl group of R¹ may beformed from one or a plurality of oxyalkyl units, such as 2, 3, 4, 5, 6,7, 8, 9, 10, or more than 10 repeating units.

In one embodiment, the present invention provides a process for formingan N-phosphoroamidate diester (110) as illustrated in Scheme 1.

In Scheme 1, a suitably protected alkyl-substituted phosphite triester(100) is reacted with an azide (105) in a solvent and in the presence ofa halide anion source such as lithium chloride to form aN-phosphoroamidate diester (110) where G¹ is H or a protecting group andR² is hydrogen.

Thus, in one embodiment, the present disclosure provides a process offorming a phosphoromonoamidate diester 110 from a phosphite triestercompound (100) and an azide compound (105),

the process comprising combining (100) with (105) in the presence of ahalide anion, such as lithium chloride, in a suitable solvent such asdimethylsulfoxide, at a suitable reaction temperature such as about 55°C., and for a suitable time period such as about 24-36 hours, wherein:

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen and C₁-C₄alkyl;

R⁴ is selected from

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R² is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom;

G² is selected from oxygen, sulfur and CH₂; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

As illustrated in Scheme 2, the protected phosphite (100) may besynthesized from the corresponding N,N-diisopropylphosphoramidite (90),where (90) may be obtained from commercial sources (for example, fromChemgenes of Wilmington, Mass., USA or Berry and Associates of Dexter,Mich., USA) or synthesized by methods known in the art. The reaction ofcompound (90) with an alcohol (HO-alkyl) in the presence of a suitableactivator such as 1H-tetrazole in a suitable solvent such asacetonitrile provides the protected phosphite (100). Activators,sometimes referred to as coupling activators, are known in the art ofphosphoramidite chemistry and oligonucleotide synthesis, where othersuitable activators include 5-ethylthio-1H-tetrazole,5-benzylthio-1H-tetrazole and 4,5-dicyanoimidazole, each available from,e.g., Glen Research (Sterling, Va.). See also, for example, Dahl, B. H.,et al. Nucleic Acids Res (1987) 15:1729-43; Vargeese, C. et al., Nucl.Acids Res. (1998) 26 (4):1046-1050; and Berner, S., Nucleic Acids Res.(1989) 17:853-64. Benzimidazolium triflate may also be used as anactivator, see, e.g., Hayakawa Y., et al., J. Org. Chem. (1996)61:7996-7997.

Thus, in another embodiment, the present disclosure provides a processwherein compound 100 is prepared from reaction of compound 90 and analcohol of formula HO-alkyl where the reaction is conducted in thepresence of an activator.

The azide (105) from Scheme 1 may be prepared from the correspondingiodo compound (85), which in turn may be prepared from the correspondingprotected hydroxyl compound (80) as illustrated in Scheme 3.

In Scheme 3, the protected hydroxyl compound (80) may be converted tothe corresponding iodo compound (85) by a two-step reaction. In thefirst step, the protecting group G³ is removed under conditions that areappropriate for that particular protecting group. For example, if theprotecting group G³ is dimethoxytrityl ether (DMTr) then G³ can beremoved by treatment with 3% trichloroacetic acid (TCA) in a suitablesolvent such as methylene chloride to provide the free hydroxylcompound, i.e., G³ is hydrogen. In the second step, the free hydroxylcompound is reacted with methyltriphenoxyphosphonium iodide in asuitable solvent such as dimethylformamide to provide the correspondingiodo compound (85). The iodo compound (85) may be readily converted tothe corresponding azide (105) by treatment with sodium azide in asuitable solvent, such as dimethylformamide.

Thus, in another embodiment, the present disclosure provides a processwherein compound 80 is converted to compound 85 and compound 85 isconverted to compound 105

where G³ is removed under conditions that are appropriate for thatparticular protecting group to provide the corresponding free hydroxylcompound, i.e., G³ is hydrogen; and the free hydroxyl compound isreacted with methyltriphenoxyphosphonium iodide in a suitable solvent toprovide the corresponding iodo compound (85), and the iodo compound (85)is converted to the corresponding azide (105) by treatment with sodiumazide in a suitable solvent.

In one embodiment, the present invention provides a process for forminga phosphate protected N-phosphoroamidate-monoester diphosphate (120) asillustrated in Scheme 4.

In Scheme 4, a suitably protected N-phosphoroamidate diester (110) isreacted with a base such as 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) inthe presence of a silylating agent such asN,O-bis-trimethylsilylacetamide (BSA) to form a first intermediate (notshown in Scheme 4) which is subsequently reacted, optionally andpreferably without isolation, with a phosphorylating agent such as thephosphorylating phosphoramidite (115, available commercially from, e.g.,Chemgenes, Wilmington, Mass., USA) as shown in Scheme 4, in the presenceof an activator such as 5-(ethylthio)-1H-tetrazole (ETT) to form asecond intermediate (not shown in Scheme 4) which is subsequentlyreacted, optionally and preferably without isolation, with an oxidizingagent such as an organic peroxide such as t-butylhydroperoxide in asuitable solvent such as methylene chloride to form the phosphateprotected N-phosphoroamidate-monoester diphosphate (120).

Thus, in one embodiment, the present disclosure provides a process offorming a phosphate protected N-phosphoroamidate-monoester disphosphate(120) from a phosphoroamidate diester compound (110) and aphosphorylating phosphoramidite compound 115,

the process comprising combining (110) with a base and a silylatingagent to provide a first intermediate, combining the first intermediatewith (115) and an activator to provide a second intermediate, andcombining the second intermediate with an oxidizing agent to form thephosphate protected N-phosphoroamidate-monoester diphosphate (120),wherein:

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen and C₁-C₄alkyl;

R⁴ is selected from

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R² is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom;

G² is selected from oxygen, sulfur and CH₂; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

In one embodiment, the present invention provides a process for forminga phosphate protected N-phosphoroamidate-monoester triphosphate (125) asillustrated in Scheme 5.

In Scheme 5, a suitably protected phosphate protectedN-phosphoroamidate-monoester diphosphate (120) is reacted with a basesuch as DBU and a silylating agent such as BSA to form a firstintermediate (not shown in Scheme 5) which is subsequently reacted,optionally and preferably without isolation, with a phosphorylatingagent such as the phosphorylating phosphoramidite (115) as shown inScheme 5, in the presence of an activator such as ETT to form a secondintermediate (not shown in Scheme 5) which is subsequently reacted,optionally and preferably without isolation, with an organic peroxidesuch as t-butylhydroperoxide in a suitable solvent such as methylenechloride to form the phosphate protected N-phosphoroamidate-monoestertriphosphate (125).

Thus, in one embodiment, the present disclosure provides a process offorming a phosphate protected N-phosphoroamidate-monoester triphosphate(125) from a phosphate protected N-phosphoroamidate-monoesterdiphosphate compound (120) and a phosphorylating phosphoramiditecompound (115),

the process comprising combining (120) with a base and a silylatingagent to provide a first intermediate, combining the first intermediatewith (115) and an activator to provide a second intermediate, andcombining the second intermediate with an oxidizing agent to form thephosphate protected N-phosphoroamidate-monoester triphosphate (125),

wherein R¹ is selected from an alkyl group and an oxyalkyl group, eitherof which terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen and C₁-C₄alkyl;

R⁴ is selected from

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R² is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R² is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom;

G² is selected from oxygen, sulfur and CH₂; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

The phosphate protected N-phosphoroamidate-monoester triphosphate (125),which may be prepared as shown in Scheme 5, comprises protecting groupsG¹, and may include a solid support SS through R⁸ of R⁴. In oneembodiment, phosphate protected N-phosphoroamidate-monoestertriphosphate (125) is exposed to conditions suitable for removing theprotecting groups G¹ and cleaving the linker of the solid support ifpresent. The choice of suitable conditions will depend on the identityof the protecting groups and the linking group that have been employedto make (125). In the case where the protecting groups are base labile,e.g., trimethylsilyl groups, and the linker is base labile, e.g., R⁸ isSS—NH—(C═O)—CH₂—O—Ar—O—CH₂C(═O)—O—, then treatment of protected andsupport-bound (125) with concentrated ammonium hydroxide at roomtemperature for about 5 minutes will release the phosphoramidite fromthe solid support and remove the protecting groups G¹. In the exemplarycase where R² is hydrogen, G² is oxygen, and R⁴ represents a cyclicsugar, these reaction conditions will provide (130),

wherein R¹ is selected from an alkyl group and an oxyalkyl group, eitherof which terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹;

R⁹ is hydrogen or, when R⁷ is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond; and

R¹¹ is hydrogen.

As described elsewhere herein, in one embodiment the present inventionprovides a process for forming a compound (130) by deprotecting acompound (125), where compound (125) may be synthesized as shown inScheme 5. In another embodiment, the present disclosure provides analternative process for forming a compound (130) which is illustrated inSchemes 6-8.

In Scheme 6, a cyclic phosphite (145) is prepared from an alcohol R¹—OH(compound 140), e.g., 5-hexyn-1-ol, and commercially available2-chloro-4H-1,3,2-benzodioxaposphorin-4-one (compound 135) by combiningthese reactants in a suitable solvent such a dimethylformamide and inthe presence of a suitable base such as tributylamine, at a suitablereaction temperature such as about room temperature, for a suitableperiod of time such as for about 5-60 minutes, to prepare compound 145,e.g., salicyl-(5-hexyn-1-yl) phosphite. Suitable reaction conditions aredisclosed in, e.g., Ludwig and Eckstein, J. Org. Chem. 56:1777-1783(1991).

The product (145) from Scheme 6 may be added to commercially available0.5M bis-tributylammonium pyrophosphate (150) in a suitable solvent,such as dimethylformamide, at a suitable temperature such as about roomtemperature, and for a suitable period of time, such as for 5-60minutes, to provide the salt of the cyclotriphosphite compounds (155),as illustrated in Scheme 7.

The cyclotriphosphite (155) may be reacted with previously describedazide (105) under suitable reaction conditions such as in a suitablesolvent such as dimethylsulfoxide, at a suitable reaction temperaturesuch as about 55° C., and for a suitable time period such as about 24-36hours, as shown in Scheme 8,

In the case were the reactions illustrated in Schemes 6, 7 and 8 areconducted on a solid support, i.e., where R⁴ includes a solid support aspart of R⁸, then the linkage to the solid support may be cleaved undersuitable reaction conditions. For example, if a base labile linker ispresent, such as when R⁸ is SS—NH—(C═O)—CH₂—O—Ar—O—CH₂C(═O)—O—, thentreatment of support-bound (160) with concentrated ammonium hydroxide atroom temperature for about 5 minutes will release the phosphoramiditefrom the solid support. In the exemplary case where R² is hydrogen, G²is oxygen, and R⁴ represents a cyclic sugar, these reaction conditionsprovide an alternative route to compound (130),

wherein R¹ is selected from an alkyl group and an oxyalkyl group, eitherof which terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹;

R⁹ is hydrogen or, when R⁷ is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond; and

R¹¹ is hydrogen.

Thus, in one embodiment the present disclosure provides a process forforming a N-phosphoroamidate-monoester triphosphate (160) from acyclotriphosphate (155) and an azide (105)

the process comprising combining (155) and (105) in the presence ofsolvent so as to form (160), wherein:

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a functional group selected from carbon-carbondouble bond, carbon-carbon triple bond, hydroxyl, amine, azide,hydrazine, thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁴ is selected from

R⁶ is a heterocycle, optionally substituted with R¹³, where R¹³ isselected from an alkyl group and an oxyalkyl group, either of whichterminates in a functional group selected from carbon-carbon doublebond, carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine,thiol, carboxyl, formyl, hydroxylamino and halogen;

R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R⁷ is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G² is selected from oxygen, sulfur and CH₂; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

In the foregoing Schemes 1-8, and within the disclosure as providedherein, the R⁸ group may be or includes a solid support, so that, forexample, one or more of, and preferably all of, the conversion ofcompound (80) to compound (85), the conversion of compound (85) to(105), the conversion of compound (105) to compound (110), and theconversion of compound (155) to compound (160) is performed using solidphase synthesis techniques.

Stratos Genomics has developed a method called Sequencing by Expansion(“SBX”) that uses a biochemical process to transcribe the sequence ofDNA onto a measurable polymer called an “Xpandomer” (Kokoris et al.,U.S. Pat. No. 7,939,259, “High Throughput Nucleic Acid Sequencing byExpansion”). The transcribed sequence is encoded along the Xpandomerbackbone in high signal-to-noise reporters that are separated by ^(˜)10nm and are designed for high-signal-to-noise, well-differentiatedresponses. These differences provide significant performanceenhancements in sequence read efficiency and accuracy of Xpandomersrelative to native DNA. Xpandomers can enable several next generationDNA sequencing detection technologies and are well suited to nanoporesequencing. The compounds as disclosed herein, and the synthetic methodsas disclosed herein, may be used in carrying out SBX.

Xpandomers are generated from polymerization of non-natural nucleotideanalogs, termed XNTPs, which are expandable, 5′ triphosphate modifiednucleotide substrates compatible with template dependent enzymaticpolymerization. An XNTP has two distinct functional regions; namely, anucleoside triphosphoramidate and a tether that is attached within eachnucleoside triphosphoramidate at positions that allow for controlledexpansion by intra-nucleotide cleavage of the phosphoramidate bond.XNTPs are described in the FIGURE in more detail.

As depicted in the FIGURE, the XNTP 100 is comprised of nucleobasetriphosphoramidate 110 with linker arm moieties 120A (which is shown asa C₄ hydrocarbon chain that is a part of R¹ as disclosed herein) and120B (which is shown as a C₆ hydrocarbon chain that is part of R¹³ asdisclosed herein) separated by selectively cleavable phosphoramidatebond 130. Each linker 120A and 120B attaches to one end of tether 140via a linking group (LG), as disclosed in U.S. Pat. No. 8,324,360 toKokoris et al., which is herein incorporated by reference in itsentirety. Tethers are polymers or molecular constructs having agenerally linear dimension and with an end moiety at each of twoopposing ends which are attached to the nucleobase triphosphoramidate110 via the reaction products of the terminal functional groups of R¹and R¹³ to form the XNTP 100. XNTPs have a “constrained configuration”and an “expanded configuration”. The constrained configuration is foundin XNTPs and in the daughter strand. The constrained configuration ofthe XNTP is the precursor to the expanded configuration, as found inXpandomer products. The transition from the constrained configuration tothe expanded configuration occurs upon scission of the P—N bond 130 ofthe phosphoramidate within the primary backbone of the daughter strand.

Tethers are joined to the nucleoside triphosphoramidate at linking group150A and 150B, wherein a first tether end is joined to the heterocycle(represented in the FIGURE by the symbol “B₁₋₄”, wherein the subscriptindicates that the heterocycle may be any one of the four standardnucleobases, A, C, G, or T) and the second tether end is joined to thealpha phosphate of the nucleobase backbone. For example, to synthesize aXATP monomer, the amino linker on 7-(octa-1,7-dinyl)-7-deaza-2′-dATP canbe used as a first tether attachment point, and a mixed backbone linker,such as the non-bridging modification (N−1-aminoalkyl) phosphoramidatecan be used as a second tether attachment point. The skilled artisanwill appreciate that many suitable coupling chemistries known in the artmay be used to form the final XNTP substrate product, for example,tether conjugation may be accomplished through a triazole linkage.

Thus, the present disclosure provides a process in which anN-phosphoroamidate-monoester triphosphate (160) as described previouslyis reacted with a tether precursor of the formula X-T-X where Xrepresents a reactive functional group that is reactive with theterminating functional groups of R¹ and R¹³, so as to form linker groupsLG1 and LG2. Optionally, X-T-X may be a bis-azide compound of theformula N₃-T-N₃, and the terminating functional groups of R¹ and R¹³ arealkyne groups, so as to form triazole groups LG1 and LG2.

After the tether has been joined to the phosphoramidate, the resultingcompound is an XNTP of the formula

wherein

R¹ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a linker group (LG1), the LG1 bonded to a tether(T);

R² is selected from hydrogen and C₁-C₄alkyl;

R³ is selected from R⁵ and —[Pn-O]_(m)—R⁵, where Pn is independentlyselected from P(OR⁵) and P(═O)(OR⁵) at each occurrence, and m isselected from 1, 2, 3, 4, 5 and 6;

R⁴ is selected from

R⁵ is selected from H and G¹;

R⁶ is a heterocycle, the heterocycle comprising a substituent R¹³, whereR¹³ is selected from an alkyl group and an oxyalkyl group, either ofwhich terminates in a linker group (LG2), the LG2 bonded to the tether(T);

R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl and—CH₂—OR¹⁰;

R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support optionallybound to a linker;

R⁹ is hydrogen or, when R⁷ is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹² whereR¹⁰ and R¹² form a direct bond;

R¹¹ is selected from H and G³;

G¹ is H or a protecting group for a hydroxyl group that is bonded to aphosphorous atom;

G² is selected from oxygen, sulfur and CH₂; and

G³ is a protecting group for a hydroxyl group that is bonded to a carbonatom.

In optional embodiments of the XNTP, which are exemplary only of XNTPembodiments as provided herein, and any of which may be combined, thepresent disclosure provides embodiments wherein:

-   -   (a) R¹ is an alkyl group which terminates in a linker group        (LG1), the LG1 bonded to a tether (T);    -   (b) R¹ is an oxyalkyl group which terminates in a linker group        (LG1), the LG1 bonded to a tether (T);    -   (c) R² is hydrogen;    -   (d) R³ is R⁵;    -   (e) R³ is —[Pn-O]_(m)—R⁵, where Pn is independently selected        from P(OR⁵) and P(═O)(OR⁵) at each occurrence, and m is selected        from 1, 2, 3, 4, 5 and 6, or m is selected from 2, 3, 4, 5 and        6; or m is 2; or m is 3; or m is 4; or m is 5; or m is 6;    -   (f) R³ is —[Pn-O]_(n)—R⁵, where Pn is P(═O)(OR⁵) at each        occurrence, and m is selected from 1, 2, 3, 4, 5 and 6, or m is        selected from 2, 3, 4, 5 and 6; or m is 2; or m is 3; or m is 4;        or m is 5; or m is 6;    -   (g) R⁴ is

-   -   (h) R⁴ is

-   -   (i) R⁵ is H;    -   (j) R⁶ is a heterocycle, the heterocycle comprising a        substituent R¹³, where R¹³ is an alkyl group which terminates in        a linker group (LG2), the LG2 bonded to the tether (T);    -   (k) R⁶ is a heterocycle, the heterocycle comprising a        substituent R¹³, where R¹³ is an oxyalkyl group which terminates        in a linker group (LG2), the LG2 bonded to the tether (T);    -   (l) R⁷ is hydrogen;    -   (m) R² is —CH₂-halogen;    -   (n) R⁷ is C₁-C₄alkyl;    -   (o) R⁷ is hydroxyl;    -   (p) R⁷ is —CH₂—OR¹⁰;    -   (q) R⁸ is —OR¹¹;    -   (r) R⁸ is —O-L-SS where L-SS represents a solid support        optionally bound to a linker;    -   (s) R⁹ is hydrogen;    -   (t) R⁹ is —CH₂—R¹² where R¹⁰ and R¹² form a direct bond;    -   (u) R¹¹ is H;    -   (v) R¹¹ is G³;    -   (w) G¹ is H;    -   (x) G¹ is a protecting group for a hydroxyl group that is bonded        to a phosphorous atom;    -   (y) G² is oxygen;    -   (z) LG1 and LG2 are each triazole.

During assembly, the monomeric XNTP substrate construct is polymerizedon the extendable terminus of the nascent daughter strand by a processof template-directed polymerization using a single-stranded template asa guide. Generally, this process is initiated from a primer and proceedsin the 5′ to 3′ direction. Generally, a DNA polymerase or otherpolymerase is used to form the daughter strand, and conditions areselected so that a complementary copy of the template strand isobtained.

As mentioned previously, further details may be found in InternationalPatent Application No. PCT/US2015/03079 and U.S. Pat. No. 8,324,360. Forexample, as explained in U.S. Pat. No. 8,324,360, a “tether” or “tethermember” refers to a polymer or molecular construct having a generallylinear dimension and with an end moiety at each of two opposing ends. Atether is attached to a substrate with a linkage in at least one endmoiety to form a substrate construct. The end moieties of the tether maybe connected to cleavable linkages to the substrate or cleavableintra-tether linkages that serve to constrain the tether in a“constrained configuration”. After the daughter strand is synthesized,each end moiety has an end linkage that couples directly or indirectlyto other tethers. The coupled tethers comprise the constrained Xpandomerthat further comprises the daughter strand. Tethers have a “constrainedconfiguration” and an “expanded configuration”. The constrainedconfiguration is found in substrate constructs and in the daughterstrand. The constrained configuration of the tether is the precursor tothe expanded configuration, as found in Xpandomer products. Thetransition from the constrained configuration to the expandedconfiguration results cleaving of selectively cleavable bonds that maybe within the primary backbone of the daughter strand or intra-tetherlinkages. A tether in a constrained configuration is also used where atether is added to form the daughter strand after assembly of the“primary backbone”. Tethers can optionally comprise one or morereporters or reporter constructs along its length that can encodesequence information of substrates. The tether provides a means toexpand the length of the Xpandomer and thereby lower the sequenceinformation linear density

“Tether constructs” are tethers or tether precursors composed of one ormore tether segments or other architectural components for assemblingtethers such as reporter constructs, or reporter precursors, includingpolymers, graft copolymers, block copolymers, affinity ligands,oligomers, haptens, aptamers, dendrimers, linkage groups or affinitybinding group (e.g., biotin).

“Tether element” or “tether segment” (T) is a polymer having a generallylinear dimension with two terminal ends, where the ends formend-linkages (LG1 and LG2) for concatenating the tether elements. Aprecursor to such a tether element may have the formula X-T-X wherein Trepresents the tether element and X is a reactive functional group thatwill react so as to form end-linkages LG1 and LG2, where LG1 and LG2 arealso joined to a nucleobase triphosphoramidate, and are shown in theFIGURE as 150B and 150A, respectively. Tether elements may be segmentsof tether constructs. Such polymers can include, but are not limited to:polyethylene glycols, polyglycols, polypyridines, polyisocyanides,polyisocyanates, poly(triarylmethyl)methacrylates, polyaldehydes,polypyrrolinones, polyureas, polyglycol phosphodiesters, polyacrylates,polymethacrylates, polyacrylamides, polyvinyl esters, polystyrenes,polyamides, polyurethanes, polycarbonates, polybutyrates,polybutadienes, polybutyrolactones, polypyrrolidinones,polyvinylphosphonates, polyacetamides, polysaccharides,polyhyaluranates, polyamides, polyimides, polyesters, polyethylenes,polypropylenes, polystyrenes, polycarbonates, polyterephthalates,polysilanes, polyurethanes, polyethers, polyamino acids, polyglycines,polyprolines, N-substituted polylysine, polypeptides, side-chainN-substituted peptides, poly-N-substituted glycine, peptoids, side-chaincarboxyl-substituted peptides, homopeptides, oligonucleotides,ribonucleic acid oligonucleotides, deoxynucleic acid oligonucleotides,oligonucleotides modified to prevent Watson-Crick base pairing,oligonucleotide analogs, polycytidylic acid, polyadenylic acid,polyuridylic acid, polythymidine, polyphosphate, polynucleotides,polyribonucleotides, polyethylene glycol-phosphodiesters, peptidepolynucleotide analogues, threosyl-polynucleotide analogues,glycol-polynucleotide analogues, morpholino-polynucleotide analogues,locked nucleotide oligomer analogues, polypeptide analogues, branchedpolymers, comb polymers, star polymers, dendritic polymers, random,gradient and block copolymers, anionic polymers, cationic polymers,polymers forming stem-loops, rigid segments and flexible segments.

Reporter element” is a signaling element, molecular complex, compound,molecule or atom that is also comprised of an associated “reporterdetection characteristic”. Other reporter elements include, but are notlimited to, FRET resonant donor or acceptor, dye, quantum dot, bead,dendrimer, upconverting fluorophore, magnet particle, electron scatterer(e.g., boron), mass, gold bead, magnetic resonance, ionizable group,polar group, hydrophobic group. Still others are fluorescent labels,such as but not limited to, ethidium bromide, SYBR Green, Texas Red,acridine orange, pyrene, 4-nitro-1,8-naphthalimide, TOTO-1, YOYO-1,cyanine 3 (Cy3), cyanine 5 (Cy5), phycoerythrin, phycocyanin,allophycocyanin, FITC, rhodamine, 5(6)-carboxyfluorescein, fluorescentproteins, DOXYL (N-oxyl-4,4-dimethyloxazolidine), PROXYL(N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO(N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, acridines,coumarins, Cy3 and Cy5 (Biological Detection Systems, Inc.), erytrosine,coumaric acid, umbelliferone, texas red rhodaine, tetramethyl rhodamin,Rox, 7-nitrobenzo-1-oxa-1-diazole (NBD), oxazole, thiazole, pyrene,fluorescein or lanthamides; also radioisotopes, ethidium, Europium,Ruthenium, and Samarium or other radioisotopes; or mass tags, such as,for example, pyrimidines modified at the C5 position or purines modifiedat the N7 position, wherein mass modifying groups can be, for examples,halogen, ether or polyether, alkyl, ester or polyester, or of thegeneral type XR, wherein X is a linking group and R is a mass-modifyinggroup, chemiluminescent labels, spin labels, enzymes (such asperoxidases, alkaline phosphatases, beta-galactosidases, and oxidases),antibody fragments, and affinity ligands (such as an oligomer, hapten,and aptamer). Association of the reporter element with the tether can becovalent or non-covalent, and direct or indirect. Representativecovalent associations include linker and zero-linker bonds. Included arebonds to the tether backbone or to a tether-bonded element such as adendrimer or sidechain. Representative non-covalent bonds includehydrogen bonds, hydrophobic bonds, ionic bonds, pi-bond ring stacking,Van der Waals interactions, and the like. Ligands, for example, areassociated by specific affinity binding with binding sites on thereporter element. Direct association can take place at the time oftether synthesis, after tether synthesis, and before or after Xpandomersynthesis.

A “reporter” is composed of one or more reporter elements. Reportersinclude what are known as “tags” and “labels.” The probe or nucleobaseresidue of the Xpandomer can be considered a reporter. Reporters serveto parse the genetic information of the target nucleic acid.

“Reporter construct” comprises one or more reporters that can produce adetectable signal(s), wherein the detectable signal(s) generally containsequence information. This signal information is termed the “reportercode” and is subsequently decoded into genetic sequence data. A reporterconstruct may also comprise tether segments or other architecturalcomponents including polymers, graft copolymers, block copolymers,affinity ligands, oligomers, haptens, aptamers, dendrimers, linkagegroups or affinity binding group (e.g., biotin).

The Examples and preparations provided below further illustrate andexemplify the compounds of the present invention and methods ofpreparing such compounds. It is to be understood that the scope of thepresent invention is not limited in any way by the scope of thefollowing Examples and preparations. In the following Examples,molecules with a single chiral center, unless otherwise noted, exist asa racemic mixture. Those molecules with two or more chiral centers,unless otherwise noted, exist as a racemic mixture of diastereomers.Single enantiomers/diastereomers may be obtained by methods known tothose skilled in the art. The starting materials and various reactantsutilized or referenced in the examples may be obtained from commercialsources, or are readily prepared from commercially available organiccompounds, using methods well-known to one skilled in the art.

EXAMPLES Materials and General Methods:

The following materials, having the abbreviations as indicated, wereobtained from the mentioned sources in the United States, unlessotherwise indicated. Aminopropyl-CPG 500A, 120-200 mesh (PrimeSynthesis, Inc., Aston, Pa.). HQDA (1) (Hydroquinone-O,O′-diacetic acid,Alfa Aesar, Ward Hill, Mass.). DMAP (4-Dimethylamino pyridine, TCIAmerica, Portland, Oreg.). Bis-CNET(Bis-cyanoethyl-N,N-diisopropylphosphoramidite, ChemGenes, Wilmington,Mass.). BSA (N,O-bis(trimethylsilyl)acetamide) (Acros Organics, NJ).HBTU (1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (EMD Millipore, Billerica, Mass.).Methyltriphenoxyphosphonium iodide (Toronto Research Chemicals, Toronto,ON CANADA). 5′-O-Dimethoxytrityl-5-(octa-1,7-diynyl)-2′-deoxyuridine (3)(ChemBiotech, Munster, Germany). 0.5M Bis-tributylammonium pyrophosphatein DMF (15, GL Synthesis Inc., Worcester, Mass.). 5′-hexynylphosphoramidite(5-hexyn-1-yl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite) fromGlen Research, Inc., Sterling, Va. ETT (5-Ethylthio-1H-tetrazole), fromGlen Research, Inc., Sterling, Va. LiCl (lithium chloride), DIEA(diisopropylethylamine), DBU (1,8-diazabicyclo[5.4.0]undec-7-ene, TBHP(t-butylhydroperoxide), DCM (dichloromethane), ACN (acetonitrile) andDMF (dimethylformamide) may each be obtained from Sigma, St. Louis, Mo.2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (salicyl chlorophosphite)(12); and 5-hexyn-1-ol (13) may also be obtained from Sigma, St. Louis,Mo. Solvents are anhydrous and packaged in SureSeal™ containers orequivalent. 2M triethylammonium acetate, Pac₂O Cap A (5% (w/v)phenoxyacetic anhydride: 10% pyridine in THF, and Pac₂O Cap B (16%1-methylimidazole in THF may each be obtained from Glen Research,Sterling, Va.

High performance liquid chromatography is performed on a ProStar Helix™HPLC system from Agilent Technologies, Inc. (Santa Clara, Calif.)consisting of two pumps (ProStar 210 Solvent Delivery Modules) with 10ml titanium pump heads, a column oven (ProStar 510 Air Oven), a UVdetector (ProStar 320 UV/Vis Detector) set at 292 nm. The system iscontrolled by Star Chromatography Workstation Software (Version 6.41).The column used is a Cadenza CD-C18, 3 μm (4.6 mm×150 mm) equipped withan in-line Cadenza Guard Column System for CD-C18 (2.0 mm×5 mm) bothfrom Imtakt USA (Portland, Oreg.). The buffers used are: Buffer A (100mM triethylammonium acetate, pH 7.0) and Buffer B (100 mMtriethylammonium acetate, pH 7.0 with 95% by volume acetonitrile).

Automated solid phase synthesis was done on a MerMade™ 12 Synthesizer(Bioautomation Corp., Plano, Tex.). Synthesis solutions for the MerMade™12 were purchased from Glen Research (Sterling, Va.).

ESI Mass spectrometry was done by Numega Resonance Lab (San Diego,Calif.). Mass specs on CPG-bound intermediates were performed on theproducts recovered after deprotection and cleavage off of the solidsupport. All ESI MS (positive mode) were consistent with the fullydeprotected structures.

Synthetic Scheme A provides an outline of a methodology according to thepresent disclosure which is described in more detail in numberedExamples 1-8. The compounds 1-10 from Scheme A were used and/orsynthesized in a glove box in a positive pressure argon atmosphere.

In Scheme A, the solid support is controlled pore glass (CPG), where CPGis an exemplary solid support of the present disclosure. Controlled poreglass (CPG) optionally, and typically does, include one or more of aplurality of reactive functional groups which may be reacted with alinking group precursor (e.g., HQDA, (1) as shown in Scheme A) toprovide compound (2). The compound (2) is then coupled, or in otherwords linked, to a precursor of the compounds of the present disclosure,in this case through the hydroxyl group of a pentose ring of (3), toprovide compound (4). Compound (4) is shown as including an exemplarysolid support SS, namely CPG, and an exemplary linking group L, in thiscase presented by “Q”, where Q representspropyl-NH—C(═O)—CH₂—O—Ar—O—CH₂—C(═O)—. Other reactive solid supportssuitable for use in the present disclosure are known to the skilledperson, and many of them are commercially available.

Example 1 HQDA-CPG (2)

HQDA-CPG (2) was prepared according to the method of Pon et. al., “RapidEsterification of Nucleosides to Solid-Phase Supports forOligonucleotide Synthesis Using Uronium and Phosphonium CouplingReagents,” Bioconjugate Chemistry, 10(6), 1051-1057 (1999).Aminopropyl-CPG (1, 1 g, 213 μmol amine) was transferred into a fritted20 mL syringe and washed with acetonitrile (3×5 mL). DMAP (65.1 mg, 532μmol) and HBTU (202 mg, 532 μmop were combined in a 8 mL polypropylenescrew capped tube and mixed with acetonitrile (5 mL). To this tube wasadded HQDA (120.4 mg, 532 μmol) and DIEA (186 μL, 1065 μmol). A chalkyprecipitate formed and was removed by centrifugation and decanting thesupernatant. The supernatant was added to the CPG in the frittedsyringe. The syringe was capped on both ends and mixed on an invertingrotator for 2 hours. The syringe was mounted on a vacuum manifoldequipped with a stopcock, drained and sequentially washed withacetonitrile (3×5 mL), methanol (2×5 mL), acetonitrile (2×5 mL) andmethylene chloride (2×5 mL) to provide the title compound (2) in pureform. Confirmation of the HQDA coupling on the CPG was based on steptrityl cation color formation as described in the following Example 2.

Example 2 5-(Octa-1,7-diynyl)-2′-deoxyuridine-3′-O-HQDA-CPG (4)

To a 8 mL polypropylene tube was added HBTU (121 mg, 319 μmol), DMAP (39mg, 319 μmop and 5′-O-dimethoxytriyl-5-(octa-1,7-diynyl)-2′-deoxyuridine(3) (216 mg, 319 μmop dissolved in acetonitrile (4.8 mL). This solutionwas added to dry HQDA-CPG (2) (1 g) in a separate polypropylene tube.The tube was capped and mixed on an inverting rotator for 20 hours atroom temperature. The slurry was transferred to a syringe equipped witha frit and stopcock. The syringe was mounted on a vacuum manifold andthe reaction flow and acetonitrile wash (2×10 mL) were collected forsubsequent recovery of uncoupled nucleoside. The solid support waswashed on the manifold with methanol (2×10 mL), acetonitrile (2×10 mL)methylene chloride (2×10 mL) and dried with vacuum.

The syringe was fitted with a closed stopcock and exposed to a solutionof 16% 1-methylimidazole in THF (Pac₂O Cap B, 5 mL) followed by asolution of 5% (w/v) phenoxyacetic anhydride: 10% pyridine in THF (Pac₂OCap A, 5 mL) was added to the dry CPG. The syringe barrel was pluggedwith a plunger and mixed on an inverting rotator for 30 minutes. Thesyringe was mounted on a vacuum manifold and washed with acetonitrile(3×10 mL), methylene chloride (2×10 mL) and dried with vacuum. The solidsupport was deblocked by flowing 3% dichloroacetic acid in methylenechloride (8×10 mL). The solid support was washed with acetonitrile untilcolorless and then washed with acetonitrile (3×10 mL) and methylenechloride (2×10 mL). After the washes, the solid support was dried on thevacuum manifold to provide (4).

Example 3 5′-Iodo-5-(Octa-1,7-diynyl)-2′,5′-dideoxyuridine-3′-O-HQDA-CPG(5)

Iodination of compound (4) was performed according to the method ofMiller and Kool, “A Simple Method for Electrophilic Functionalization ofDNA,” Org. Lett., 4(21),3599-3601(2002). Solid support bound5-(octa-1,7-diynyl)-2′-deoxyuridine-3′-O-HQDA-CPG (4) (330 mg) wastransferred to a syringe fitted with a stopcock and loaded onto a vacuummanifold. The solid support was wetted with DMF (1×10 mL). Freshlyprepared 0.5 M methyltriphenoxyphosphonium iodide in DMF (10 mL) wasadded to the syringe, the barrel was capped and the contents were mixedon an inverting rotator for 1 hour. The syringe was fitted on a vacuummanifold and washed with DMF (4×10 mL), acetonitrile (3×10 mL) andmethylene chloride (3×10 mL). The solid support was dried with viavacuum to provide purified (5).

Example 45′-Azido-5-(Octa-1,7-diynyl)-2′,5′-dideoxyuridine-3′-O-HQDA-CPG (6)

Sodium azide (130 mg, 20 mmol) and sodium iodide (300 mg, 20 mmol) weredissolved in DMF (20 mL) in an amber glass vial. A molecular sievepacket was added to the solution and let stand overnight. The next day,the 100 mmol azide/iodide solution (10 mL) was added to solid supportbound 5′-iodo-5-(octa-1,7-diynyl)-2′,5′-dideoxyuridine-3′-O-HQDA-CPG (5)(330 mg) in a 15 mL polypropylene tube. The solution was incubated at50° C. for 3 hours with no agitation. The solid support was rinsed withDMF, centrifuged, and the DMF supernatant was decanted. The solidsupport was slurried with fresh DMF and transferred to a frittedsyringe. The solid support was washed with DMF (3×10 mL), acetonitrile(3×10 mL) and methylene chloride (2×10 mL). The solid support was driedto a free flowing powder (6) on the vacuum manifold. See, e.g., Millerand Kool, “Versatile 5′-Functionalization on Solid Support: Amines,Azides, Thiols and Thioethers via Phosphorus Chemistry,” J. OrganicChemistry, 69(7), 2404-2410 (2004).

A MS sample was prepared by transferring a small amount of (6) to a 2 mLscrew cap tube. Cold NH₄OH (500 μL) was added to the solid support andthen incubated at room temperature for 5 minutes. The CPG/NH₄OH slurrywas transferred to a 3 mL syringe fitted with a 13 mm syringe filterwith a 0.45 μm GHP Acrodisc filter (Pall Corporation, Ft. Washington,N.Y.). The plunger was fitted into the syringe barrel and the filtratewas collected into a 1.5 ml polypropylene tube. The CPG/Acrodisc werewashed with cold NH₄OH (500 μL) followed by H₂O (500 μL) and added tothe original filtrate. This solution was evaporated in a Savant Speedvacat 65° C. for 1 hour followed by evaporation at room temperature toreduce the volume to at least 150 μL. The crude material HPLC purifiedwith a Cadenza CD-C18 column (4.6 mm×150 mm 3 μM) on the Prostar Systemusing a gradient of 5% B to 39.5% B in 46 minutes at 1 mL/min andmonitoring at 292 nm. The peak containing the 5′-azide (6) was sent toNumega Resonance Labs (San Diego, Calif.) for ESI MS analysis. The foundm/z was in agreement with the calculated m/z for the structure shownbelow.

Example 5 5-Hexyn-1-yl-(2-Cyanoethyl)-methyl-Phosphite (7)

Phosphite (7) was prepared by dissolving5-hexyn-1-yl-(2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite (403μmole) in dry acetonitrile (4 mL) with 0.28 M 5-ethylthio-1H-tetrazole(1.726 mL, 84 mmol). Methanol (28 μL, 119 mmol) was added and sharpneedles of N,N-diisopropylammonium ethylthiotetrazolide formedimmediately. The solution was incubated at room temperature for 2 hours.The solution was separated from the crystals and the supernatant wasdivided into 4 polypropylene tubes and evaporated to form a solid mass(7).

Example 65′-N-(2-Cyanoethyl)-(5-Hexyn-1-yl)-Phosphoramidate-5-(Octa-1,7-diynyl)-2′,5′-dideoxyuridine-3′-O-HQDA-CPG(8)

Solid support bound5′-azido-5-(octa-1,7-diynyl)-2′,5′-dideoxyuridine-3′-O-HQDA-CPG (6) (2.4μmols/mg, 125 mg) was transferred to a fritted syringe and mounted on avacuum manifold. The support was washed with acetonitrile (3×1 ml). Thesupport was dried in vacuo and transferred to a polypropylene tube. Tothe tube was added 0.7M solution of5-hexyn-1-yl-(2-cyanoethyl)-methyl-phosphite (7) in DMSO (429 μL, 300μmol) and a 2.5 M solution of LiCl in DMSO (300 μL, 750 umol). The tubewas capped and placed on a heated mixer and set to 55° C. and 400 rpmfor 24 hours followed by mixing at room temperature for 12 hours. Thetube was centrifuged to pellet the solid support. The solid support wastransferred to a fritted syringe, mounted on a vacuum manifold andwashed with DMF (3×1 mL), acetonitrile (2×1 mL), H₂O (1×1 mL) andacetonitrile (3×1 mL). The solid (8) was dried by vacuum on themanifold.

A MS sample was prepared by transferring a small amount of (8) to a 2 mLscrew cap tube. Cold NH₄OH (500 μL) was added to the solid support andthen incubated at room temperature for 5 minutes. The CPG/NH₄OH slurrywas transferred to a 3 mL syringe fitted with a 13 mm syringe filterwith a 0.45 μm GHP Acrodisc filter (Pall Corporation, Ft. Washington,N.Y.). The plunger was fitted into the syringe barrel and the filtratewas collected into a 1.5 ml polypropylene tube. The CPG/Acrodisc werewashed with cold NH₄OH (500 μL) followed by H₂O (500 μL) and added tothe original filtrate. This solution was evaporated in a Savant Speedvacat 65° C. for 1 hour followed by evaporation at room temperature toreduce the volume to at least 150 μL. The crude material HPLC purifiedwith a Cadenza CD-C18 column (4.6 mm×150 mm 3 μM) on the Prostar Systemusing a gradient of 5% B to 39.5% B in 46 minutes at 1 mL/min andmonitoring at 292 nm. The peak containing the (8) was sent to NumegaResonance Labs (San Diego, Calif.) for ESI MS analysis. The found m/zwas in agreement with the calculated m/z for the structure shown below.

Example 7 Protected 5′-TriphosphoramidateP_(α)-(5-Hexyn-1-yl)-5-(octa-1,7-diynyl)-2′,5′-dideoxyuridine-3′-O-HQDA-CPG(10)

The diphosphate (9) and triphosphate (10) were prepared from5′-N-(2-Cyanoethyl)-(5-hexyn-1-yl)-phosphoramidate-5-(octa-1,7-diynyl)-2′,5′-dideoxyuridine-3′-O-HQDA-CPG(8) (125 mg) on solid support using a MerMade 12 synthesizer. Thesynthesis was performed with the following 2 basic automated steps: (R)Removal of the cyanoethyl phosphate protecting group; (C) Coupling ofthe Bis-CNET and oxidation to P(V) phosphate. The sequence of additionsand delivery volumes for these routines are set forth below:

Removal of cyanoethyl protecting group (R) was performed as summarizedin Table 1.

TABLE 1 # uL per Total Additions Reagent Step Addition uL 3 10% DBU/45%BSA Deprotection 250 750 in ACN 1 — Drain — — 3 10% DBU/45% BSADeprotection 250 750 in ACN 1 — Drain — — 3 10% DBU/45% BSA Deprotection250 750 in ACN 2 — Drain — — 6 ACN Wash 300 1800  1 — Drain — — 4 DCMWash 250 1000  3 — Drain — —

Coupling of Bis-CNET and oxidation to P(V) phosphate (C) was done assummarized in Table 2.

TABLE 2 # uL per Total Additions Reagent Step Addition uL 3 Bis-CNEt PPACoupling  95 285 ETT Activator 110 330 1 ACN Wash 300 300 1 — Drain — —3 1.1M t-BuOOH Oxidation 200 600 1 — Drain — — 3 DCM Wash 250 750 1 —Drain — — 3 ACN Wash 300 900 2 — Drain — —

The order of the automated steps used to make the triphosphoramidate aresummarized in Table 3.

TABLE 3 Command Treatment R Remove α phosphate cnet C Couple and oxidizeβ phosphate R Remove β phosphate cnet C Couple and oxidize γ phosphate RRemove γ phosphate cnet

Example 8 5′-TriphosphoramidateP_(α)-(5-Hexyn-1-yl)-5-(octa-1,7-diynyl)-2′,5′-dideoxyuridine (11)

Solid support bound 5′-triphosphoramidatePa-(5-hexyn-1-yl)-5-(octa-1,7-diynyl)-2′,5′-dideoxyuridine-3′-O-HQDA-CPG(10) (125 mg) was weighed into a 2 mL polypropylene tube. Cold NH₄OH(500 μL) was added to the solid support and then incubated at roomtemperature for 5 minutes. The CPG/NH₄OH slurry was transferred to a 3mL syringe fitted with a 13 mm syringe filter with a 0.45 μm GHPAcrodisc filter (Pall Corporation, Ft. Washington, N.Y.). The plungerwas fitted into the syringe barrel and the filtrate was collected into a1.5 ml polypropylene tube. The CPG/Acrodisc were washed with cold NH₄OH(500 μL) followed by H₂O (500 μL) and added to the original filtrate.This solution was evaporated in a Savant Speedvac at 65° C. for 1 hourfollowed by evaporation at room temperature to reduce the volume to atleast 150 μL. The crude material (11) was quantified by UV.

HPLC purification was performed on a Cadenza CD-C18 column (4.6 mm×150mm 3 μM) on the Prostar System using a gradient of 5% B to 39.5% B in 46minutes at 1 mL/min and monitoring at 292 nm. The peak containing thetriphosphoramidate (11) was sent to Numega Resonance Labs (San Diego,Calif.) for ESI MS analysis. Calculated m/z: 651.44 amu. Found: 650 amu(M-H).

In Example 2, 5′-O-dimethoxytriyl-5-(octa-1,7-diynyl)-2′-deoxyuridine(3) was employed as a starting material, so that the subsequently formedcompounds 4-11 each contained the uracil nucleobase. This same syntheticroute may be employed with suitable alternative octadiynyl2′-deoxynucleosides to (3) so as to incorporate alternative nucleobasesinto a compound of Formula 1 as disclosed herein. For example,N6-protected 5′-DMT-2′-deoxy-7-octadiynyl-7-deazaadenosine,N4-protected-5-octadiynyl-5′-DMT-2′-deoxycytidine, andN2-protected-5′-DMT-2′-deoxy-7-octadiynyl-7-deazaguanosine may be usedin lieu of the 5-octadiynyl-5′-DMT-2′-deoxyuridine (3).

Synthetic Scheme B provides an outline of a methodology according to thepresent disclosure which is described in more detail in numberedExamples 9-12. The compounds 6 and 11-16 from Scheme B were used and/orsynthesized in a glove box in a positive pressure argon atmosphere.

Example 9 Salicyl-(5-Hexyn-1-yl)-phosphite (14)

In a polypropylene tube, 5-hexyn-1-ol (13) (25 μL, 220 μmol) was addedto tributylamine (95 μL, 400 μmol) in DMF (343 μL). In a separatepolypropylene tube, 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one (12)(81 mg, 400 μmol) was dissolved in DMF (200 μL). See, e.g., Ludwig andEckstein, “Synthesis of Nucleoside 5′-O-(1,3-Dithiotriphosphates) and5′-O-(1,1-Dithiotriphosphates)”, J. Org. Chem, 56, 1777-1783 (1991). Thetwo DMF solutions were mixed and incubated at room temperature for 30minutes to provide salicyl-(5-hexyn-1-yl)-phosphite (14), which was useddirectly in the process described in Example 10.

Example 10 P¹-(5-Hexyn-1-yl)-P²,P³-dioxo-cyclotriphosphite (16)

Salicyl-(5-hexyn-1-yl)-phosphite (14) solution from Example 9 was addedto a mixture of tributylamine (95 μmol, 400 μmol) in 0.5Mbis-tributylammonium pyrophosphate (15) in DMF (440 μL, 220 μmol) andthe solution was incubated at room temperature for 90 minutes to providethe cyclic triphosphite (16). See, e.g., Ludwig and Eckstein, “Synthesisof Nucleoside 5′-O-(1,3-Dithiotriphosphates) and5′-O-(1,1-Dithiotriphosphates)”, J. Org. Chem, 56, 1777-1783 (1991). Theresulting solution was used in Example 11.

Example 11 5′-TriphosphoramidateP_(α)-(5-Hexyn-1-yl)-5-(octa-1,7-diynyl)-2′,5′-dideoxyuridine-3′-O-HQDA-CPG(17)

5′-Azido-5-(octa-1,7-diynyl)-2′,5′-dideoxyuridine-3′-O-HQDA-CPG (6) (9mg) was mixed with the solution containing compound 16 from Example 10,and the slurry was incubated at room temperature for 24 hours to provide5′-triphosphoramidatePa-(5-hexyn-1-yl)-5-(octa-1,7-diynyl)-2′,5′-dideoxyuridine-3′-O-HQDA-CPG(17). The slurry was transferred to a fritted Luer tip column(Bioautomation, Plano, Tex.) and fitted onto a vacuum manifold. Thereaction solution was drained and the CPG-bound derivative (17) waswashed with DMF (1×500 μL) followed by more DMF (2×1 mL) and then ACN(2×1 mL). The CPG derivative (17) was dried by vacuum on the manifold.

Example 12 5′-TriphosphoramidateP_(α)-(5-Hexyn-1-yl)-5-(octa-1,7-diynyl)-2′,5′-dideoxyuridine (11)

Solid support bound 5′-triphosphoramidatePa-(5-hexyn-1-yl)-5-(octa-1,7-diynyl)-2′,5′-dideoxyuridine-3′-O-HQDA-CPG(17) was deprotected and released from the solid support according tothe method of Example 8 with cold NH₄OH to provide 5′-triphosphoramidatePa-(5-hexyn-1-yl)-5-(octa-1,7-diynyl)-2′,5′-dideoxyuridine (11).

The skilled person may refer to one or more of the following documentsfor additional information regarding the identification and syntheticmethods that may be applied to the preparation of the compounds andprecursors thereof, of the present disclosure. Synthesis ofphosphoromonoamidate diesters (Staudinger Reaction followed byMichaelis-Arbuzov Reaction) is discussed in, e.g., Letsinger andHeavner, “Synthesis of Phosphoromonoamidate Diester Nucleotides via thePhosphite-Azide Coupling Method,” Tetrahedron Letters, 16(2), 147-150(1975). The identification and synthesis of LNA nucleosides is discussedin, e.g., Wengel et al., “LNA (Locked Nucleic Acids): Synthesis of theadenine, cytosine, guanine, 5-methylcytosine, thymine, andbicyclonucleoside monomers, oligomerisation, and unprecedented nucleicacid recognition,” Tetrahedron, 54(12), 3607-3630 (1998). Identificationand synthesis of acyclic nucleosides is discussed in, e.g., Wengel etal., “UNA (unlocked nucleic acid): A flexible RNA mimic that allowsengineering of nucleic acid duplex stability,” Bioorganic & MedicinalChemistry, 17(15), 5420-5425 (2009). The identification and use ofphosphate and other protecting groups is discussed in, e.g., Peter G. M.Wuts, “Greene's Protective Groups in Organic Synthesis: Fifth Edition,Wiley, 2014.

Although any methods and materials similar or equivalent to thosedescribed herein can also be used in the practice of the presentinvention, a limited number of the exemplary methods and materials havebeen illustrated in detail.

The present disclosure provides the following numbered embodiments,which are exemplary only of the embodiments of the present invention:

1) A compound of the formula

-   -   wherein    -   R¹ is selected from        -   a) an alkyl group and an oxyalkyl group, either of which            terminates in a functional group selected from carbon-carbon            double bond, carbon-carbon triple bond, hydroxyl, amine,            azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and            halogen; and        -   b) an alkyl group and an oxyalkyl group, either of which            terminates in a linker group (LG1), the LG1 bonded to a            tether (T);    -   R² is selected from hydrogen and C₁-C₄alkyl;    -   R³ is selected from R⁵ and —[Pn-O]_(m)—R⁵, where Pn is        independently selected from P(OR⁵) and P(═O)(OR⁵) at each        occurrence, and m is selected from 1, 2, 3, 4, 5 and 6;    -   R⁴ is selected from

-   -   R⁵ is selected from H and G¹;    -   R⁶ is a heterocycle, the heterocycle optionally comprising a        substituent R¹³, where R¹³ is selected from        -   a) an alkyl group and an oxyalkyl group, either of which            terminates in a functional group selected from carbon-carbon            double bond, carbon-carbon triple bond, hydroxyl, amine,            azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and            halogen; and        -   b) an alkyl group and an oxyalkyl group, either of which            terminates in a linker group (LG2), the LG2 bonded to the            tether (T);    -   R² is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl        and —CH₂—OR¹⁰;    -   R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support        optionally bound to a linker;    -   R⁹ is hydrogen or, when R² is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹²        where R¹⁰ and R¹² form a direct bond;    -   R¹¹ is selected from H and G³;    -   G¹ is H or a protecting group for a hydroxyl group that is        bonded to a phosphorous atom;    -   G² is selected from oxygen, sulfur and CH₂; and    -   G³ is a protecting group for a hydroxyl group that is bonded to        a carbon atom.

2) The compound of embodiment 1 wherein each of R¹ and R¹³ is selectedfrom an alkyl group and an oxyalkyl group, either of which terminates ina functional group selected from carbon-carbon double bond,carbon-carbon triple bond, hydroxyl, amine, azide, hydrazine, thiol,carboxyl, formyl, hydroxylamino and halogen.

3) The compound of embodiment 2 wherein R¹ is —(CH₂)_(q)—C≡CH, R¹³ is—C≡C—(CH₂)_(q)—C≡CH and q is an integer selected from 2-10.

4) The compound of embodiment 1 wherein each of R¹ and R¹³ is selectedfrom an alkyl group and an oxyalkyl group, either of which terminates ina linker group (LG1), the LG1 bonded to a tether (T).

5) The compound of embodiment 4 wherein LG1 and LG2 are triazole groups.

6) The compound of each of embodiments 1, 2, 3, 4 and 5 wherein R³ isselected from

7) The compound of each of embodiments 1, 2, 3, 4, 5 and 6 wherein R⁴ is

8) The compound of embodiment 1 wherein R⁶ is selected from:

-   -   an adenosine analog of formula

-   -   a guanosine analog of formula

-   -   a uridine analog of formula and

-   -   a cytidine analog of formula

-   -   and wherein R¹³ is selected from        -   a) an alkyl group and an oxyalkyl group, either of which            terminates in a functional group selected from carbon-carbon            double bond, carbon-carbon triple bond, hydroxyl, amine,            azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and            halogen; and        -   b) an alkyl group and an oxyalkyl group, either of which            terminates in a linker group (LG2), the LG2 bonded to the            tether (T).

9) The compound of embodiment 1 having the formula

wherein:

-   -   G¹ is H or a protecting group;    -   R⁶ is a heterocycle comprising a substituent R¹³;    -   R⁸ is selected from OR¹¹ and O-L-SS where SS represents a solid        support and L represents a linking group between O and the SS;    -   R¹¹ is selected from H and G³; and    -   G³ is a protecting group for a hydroxyl group that is bonded to        a carbon atom.

10) The compound of embodiment 1 having the formula

wherein:

-   -   R⁶ is a heterocycle comprising a substituent R¹³;    -   R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support        bound to a linker;    -   R¹¹ is selected from H and G³; and    -   G³ is a protecting group for a hydroxyl group that is bonded to        a carbon atom.

11) The compound of embodiment 1 having a formula selected from thegroup

wherein:

-   -   R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support        bound to a linker;    -   R¹¹ is selected from H and G³; and    -   G³ is a protecting group for a hydroxyl group that is bonded to        a carbon atom.

12) The compound of embodiment 1 wherein each of LG1 and LG2 is atriazole group.

13) A process of forming a phosphoromonoamidate diester 110 from aphosphite triester compound (100) and an azide compound (105),

-   -   the process comprising combining (100) with (105) in the        presence of a halide anion, wherein:    -   R¹ is selected from an alkyl group and an oxyalkyl group, either        of which terminates in a functional group selected from        carbon-carbon double bond, carbon-carbon triple bond, hydroxyl,        amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino        and halogen;    -   R² is selected from hydrogen and C₁-C₄alkyl;    -   R⁴ is selected from

-   -   R⁶ is a heterocycle, the heterocycle optionally comprising a        substituent R¹³, where R¹³ is selected from        -   a) an alkyl group and an oxyalkyl group, either of which            terminates in a functional group selected from carbon-carbon            double bond, carbon-carbon triple bond, hydroxyl, amine,            azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and            halogen; and        -   b) an alkyl group and an oxyalkyl group, either of which            terminates in a linker group (LG2), the LG2 bonded to the            tether (T);    -   R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl        and —CH₂—OR¹⁰;    -   R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support        bound to a linker (L);    -   R⁹ is hydrogen or, when R⁷ is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹²        where R¹⁰ and R¹² form a direct bond;    -   R¹¹ is selected from H and G³;    -   G¹ is H or a protecting group for a hydroxyl group that is        bonded to a phosphorous atom;    -   G² is selected from oxygen, sulfur and CH₂; and    -   G³ is a protecting group for a hydroxyl group that is bonded to        a carbon atom.

14) A process for forming a phosphate protectedN-phosphoroamidate-monoester disphosphate (120) from a phosphoroamidatediester compound (110) and a phosphorylating phosphoramidite compound115,

-   -   the process comprising combining (110) with a base and a        silylating agent to provide a first intermediate, combining the        first intermediate with (115) and an activator to provide a        second intermediate, and combining the second intermediate with        an oxidizing agent to form the phosphate protected        N-phosphoroamidate-monoester diiphosphate (120), wherein:    -   R¹ is selected from an alkyl group and an oxyalkyl group, either        of which terminates in a functional group selected from        carbon-carbon double bond, carbon-carbon triple bond, hydroxyl,        amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino        and halogen;    -   R² is selected from hydrogen and C₁-C₄alkyl;    -   R⁴ is selected from

-   -   R⁶ is a heterocycle, the heterocycle optionally comprising a        substituent R¹³, where R¹³ is selected from        -   a) an alkyl group and an oxyalkyl group, either of which            terminates in a functional group selected from carbon-carbon            double bond, carbon-carbon triple bond, hydroxyl, amine,            azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and            halogen; and        -   b) an alkyl group and an oxyalkyl group, either of which            terminates in a linker group (LG2), the LG2 bonded to the            tether (T);    -   R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl        and —CH₂—OR¹⁰;    -   R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support        optionally bound to a linker (L);    -   R⁹ is hydrogen or, when R⁷ is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹²        where R¹⁰ and R¹² form a direct bond;    -   R¹¹ is selected from H and G³;    -   G¹ is H or a protecting group for a hydroxyl group that is        bonded to a phosphorous atom;    -   G² is selected from oxygen, sulfur and CH₂; and    -   G³ is a protecting group for a hydroxyl group that is bonded to        a carbon atom.

15) A process for forming a phosphate protectedN-phosphoroamidate-monoester triphosphate (125) from a phosphateprotected N-phosphoroamidate-monoester diphosphate compound (120) and aphosphorylating phosphoramidite compound (115),

-   -   the process comprising combining (120) with a base and a        silylating agent to provide a first intermediate, combining the        first intermediate with (115) and an activator to provide a        second intermediate, and combining the second intermediate with        an oxidizing agent to form the phosphate protected        N-phosphoroamidate-monoester triphosphate (125), wherein:    -   R¹ is selected from an alkyl group and an oxyalkyl group, either        of which terminates in a functional group selected from        carbon-carbon double bond, carbon-carbon triple bond, hydroxyl,        amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino        and halogen;    -   R² is selected from hydrogen and C₁-C₄alkyl;    -   R⁴ is selected from

-   -   R⁶ is a heterocycle, the heterocycle optionally comprising a        substituent R¹³, where R¹³ is selected from        -   a) an alkyl group and an oxyalkyl group, either of which            terminates in a functional group selected from carbon-carbon            double bond, carbon-carbon triple bond, hydroxyl, amine,            azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and            halogen; and        -   b) an alkyl group and an oxyalkyl group, either of which            terminates in a linker group (LG2), the LG2 bonded to the            tether (T);    -   R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl        and —CH₂—OR¹⁰;    -   R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support        optionally bound to a linker (L);    -   R⁹ is hydrogen or, when R⁷ is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹²        where R¹⁰ and R¹² form a direct bond;    -   R¹¹ is selected from H and G³;    -   G¹ is H or a protecting group for a hydroxyl group that is        bonded to a phosphorous atom;    -   G² is selected from oxygen, sulfur and CH₂; and    -   G³ is a protecting group for a hydroxyl group that is bonded to        a carbon atom.

16) A process for forming a N-phosphoroamidate-monoester triphosphate(160) from a cyclotriphosphite (155) and an azide (105)

-   -   the process comprising combining (155) and (105) in the presence        of solvent so as to form (160), wherein:    -   R¹ is selected from an alkyl group and an oxyalkyl group, either        of which terminates in a functional group selected from        carbon-carbon double bond, carbon-carbon triple bond, hydroxyl,        amine, azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino        and halogen;    -   R⁴ is selected from

-   -   R⁶ is a heterocycle, the heterocycle optionally comprising a        substituent R¹³, where R¹³ is selected from        -   a) an alkyl group and an oxyalkyl group, either of which            terminates in a functional group selected from carbon-carbon            double bond, carbon-carbon triple bond, hydroxyl, amine,            azide, hydrazine, thiol, carboxyl, formyl, hydroxylamino and            halogen; and        -   b) an alkyl group and an oxyalkyl group, either of which            terminates in a linker group (LG2), the LG2 bonded to the            tether (T);    -   R⁷ is selected from hydrogen, —CH₂-halogen, C₁-C₄alkyl, hydroxyl        and —CH₂—OR¹⁰;    -   R⁸ is —OR¹¹ or —O-L-SS where L-SS represents a solid support        optionally bound to a linker;    -   R⁹ is hydrogen or, when R⁷ is —CH₂—OR¹⁰ then R⁹ may be —CH₂—R¹²        where R¹⁰ and R¹² form a direct bond;    -   R¹¹ is selected from H and G³;    -   G² is selected from oxygen, sulfur and CH₂; and    -   G³ is a protecting group for a hydroxyl group that is bonded to        a carbon atom.

17) The process of embodiment 16 further comprising reacting theN-phosphoroamidate-monoester triphosphate (160) with a tether precursorof the formula X-T-X where X represents a reactive functional group thatis reactive with the terminating functional group of R¹ and R¹³, so asto form linker groups LG1 and LG2.

18) The process of embodiment 17 wherein X is an azide group and theterminating functional groups of R¹ and R¹³ are alkyne groups.

19) A cyclic phosphite of the formula

-   -   wherein R¹ is selected from an alkyl group and an oxyalkyl        group, either of which terminates in a functional group selected        from carbon-carbon double bond, carbon-carbon triple bond,        hydroxyl, amine, azide, hydrazine, thiol, carboxyl, formyl,        hydroxylamino and halogen.

20) The cyclic phosphite of embodiment 20 wherein R¹ is a terminallyfunctionalized alkyl group, where the functional group is acarbon-carbon triple bond.

Where a range of values is provided herein, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges is also encompassed within the invention, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the invention.

For example, any concentration range, percentage range, ratio range, orinteger range provided herein is to be understood to include the valueof any integer within the recited range and, when appropriate, fractionsthereof (such as one tenth and one hundredth of an integer), unlessotherwise indicated. Also, any number range recited herein relating toany physical feature, such as polymer subunits, size or thickness, areto be understood to include any integer within the recited range, unlessotherwise indicated. As used herein, the term “about” means±20% of theindicated range, value, or structure, unless otherwise indicated.

All of the U.S. patents, U.S. patent application publications, U.S.patent applications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, including but not limited to U.S. Pat.Nos. 8,586,301 and 8,592,182 as well as US Patent Publication Nos.2014/134618 and 2015/0284787, and U.S. Provisional Application No.62/082,488 are incorporated herein by reference, in their entirety. Suchdocuments may be incorporated by reference for the purpose of describingand disclosing, for example, materials and methodologies described inthe publications, which might be used in connection with the presentlydescribed invention. The publications discussed above and throughout thetext are provided solely for their disclosure prior to the filing dateof the present application. Nothing herein is to be construed as anadmission that the inventors are not entitled to antedate any referencedpublication by virtue of prior invention.

In general, in the following claims, the terms used should not beconstrued to limit the claims to the specific embodiments disclosed inthe specification and the claims, but should be construed to include allpossible embodiments along with the full scope of equivalents to whichsuch claims are entitled. Accordingly, the claims are not limited by thedisclosure.

However, all structures encompassed within a claim are “chemicallyfeasible”, by which is meant that the structure depicted by anycombination or subcombination of optional substituents meant to berecited by the claim is physically capable of existence with at leastsome stability as can be determined by the laws of structural chemistryand by experimentation. Structures that are not chemically feasible arenot within a claimed set of compounds.

1-20. (canceled)
 21. A compound of the formula:

wherein R³ is R⁵,

R⁵ is H; R⁶ is: an adenosine analog of formula

a guanosine analog of formula

a uridine analog of formula or

a cytidine analog of formula

R⁸ is —OR¹¹; R¹¹ is H; and R¹ and R¹³ are taken together to form atether segment which allows for linear expansion of the compound uponcleavage of the phosphoramidate bond.
 22. The compound of claim 21wherein R³ is R⁵.
 23. The compound of claim 21 wherein R³ is


24. The compound of claim 21 wherein R³ is


25. The compound of claim 21 wherein R³ is


26. The compound of claim 21 wherein R³ is


27. The compound of claim 21 wherein R⁶ is an adenosine analog offormula


28. The compound of claim 21 wherein R⁶ is an a guanosine analog offormula


29. The compound of claim 21 wherein R⁶ is an a uridine analog offormula


30. The compound of claim 21 wherein R⁶ is an a cytidine analog offormula


31. The compound of claim 21 wherein the tether segment is attached atthe R¹ position, the R¹³ position, or both the R¹ and R¹³ positions by alinkage comprising a triazole group.
 32. The compound of claim 21wherein the tether segment comprises a reporter construct.